Bifunctional linking compounds, conjugates and methods for their production

ABSTRACT

The present invention provides novel N-substituted hydrazine bifunctional compounds, novel N-subhstituted hydrazone derivatives of a cytotoxic reagent incorporating the bifunctional compounds, novel conjugates containing at least one cytotoxic reagent molecule reacted with the bifunctional compound and bound to a molecule reactive with a target cell population, methods for their production, and pharmaceutical compositions and methods for delivering cytotoxic reagents to a target population of cells. The hydrazone bonds of the conjugates of the invention permit the release of free cytotoxic reagent from the conjugates in the acidic external or internal environment of the target cells. The bifunctional compounds, derivatives, conjugates and methods of the invention are useful in antibody-or ligand-mediated drug delivery systems for the perferential killing of a target cell population to treat diseases such as cancers, infections and autoimmune disorders.

FIELD OF THE INVENTION

The present invention relates to novel bifunctional compounds,conjugates containing the compounds and methods for their production anduse. More particularly, the invention relates to N-substituted hydrazinecompounds that may be linked to molecules for targeting cellpopulations.

BACKGROUND OF THE INVENTION

Bifunctional compounds that permit the linkage of two or more moleculeshave been described. For example, bifunctional compounds for linkingcytotoxic reagents to molecules for targeting cell populations areknown. The bifunctional compounds must be capable of carrying andreleasing these types of cytotoxic reagents in vivo, for example toprovide sufficient, i.e. therapeutic, levels of the reagents in vivo,without damaging the activity of the targeting molecules. For certainapplications, the formation of a conjugate containing a pH sensitivelinkage between the reagent and targeting molecules of the conjugateproviding release of the cytotoxic reagent in certain ranges of pH, isdesirable.

Particularly useful reagents for treatment of cancers are theanthracyclines. Anthracyclines are antibiotic compounds that exhibitcytotoxic activity. Studies have indicated that anthracyclines mayoperate to kill cells by a number of different mechanisms including: 1)intercalation of the drug molecules into the DNA of a cell therebyinhibiting DNA-dependent nucleic acid synthesis; 2) production by thedrug of free radicals which then react with cellular macromolecules tocause damage to the cells, or 3) interactions of the drug molecules withthe cell membrane (Peterson et al., "Transport And Storage OfAnthracyclines In Experimental Systems And Human Leukemia", inAnthracycline Antibiotics In Cancer Therapy, Muggia et al. (Eds.), p.132 (Martinus Nijhoff Publishers (1982); and Bachur, "Free RadicalDamage", id. at pp. 97-102)). Because of their cytotoxic potential,anthracyclines have been used in the treatment of numerous cancers suchas leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma,and sarcomas (Wiernik, "Current Status Of Adriamycin And Daunomycin InCancer Treatment", in Anthracyclines: Current Status And NewDevelopments, Crooke et al. (Eds.), pp. 273-94 (Academic Press 1980)).Commonly used anthracyclines include adriamycin (ADM also known asdoxorubicin) and daunomycin (DAU also known as daunorubicin).

Although these compounds may be useful in the treatment of neoplasms andother disease states wherein a target cell population is sought to bereduced or eliminated, their therapeutic efficacy is often limited bythe dose-dependent toxicity associated with their administration. Forexample, in the treatment of tumors, typical adverse side effects ofthese compounds include myelosuppression and cardiotoxicity (Crooke,"Goals For Anthracycline Analog Development At Bristol Laboratories",Anthracyclines: Current Status And New Developments, supra, at p. 11).Attempts have therefore been made in the treatment of tumors to improvethe therapeutic effects of these compounds by linking the anthracyclineto antibodies directed against tumor-associated antigens to formimmunoconjugates for selective delivery of the drugs to tumor cells.(Hermentin and Seiler, "Investigations with monoclonal antibody drug(anthracycline) conjugates", Behring Insti. Mitl. 82:197-215 (1988)). Inthis way, the drug can be delivered or "targeted" to the tumor site andits toxic side effects on normal cells in the body may be diminished.Immunoconjugates comprised of the anthracyclines ADM or DAU linked topolyclonal or monoclonal antibodies to tumor-associated antigens areknown in the art (e.g. Gallego et al., "Preparation Of FourDaunomucin-Monoclonal Antibody 791IT/36 Conjugates With Anti-TumorActivity", Int. J. Cancer 33: 737-44 (1984); and Arnon et al., "In VitroAnd In Vitro Efficacy Of Conjugates Of Daunomycin With Anti-TumorAntibodies", Immunological Rev. 62:5-27 (1982)).

The most frequently used approaches for the attachment of ananthracycline to an antibody have utilized a linkage at the amino sugarmoiety of the anthracycline. For example, the amino sugar has beenoxidized by sodium periodate treatment and directly attached to lysineresidues on the antibody via Schiff base formation (Hurwitz et al., "TheCovalent Binding Of Daunomycin And Adriamycin To Antibodies, WithRetention of Both Drug And Antibody Activities", Cancer Res.35:1175-1181 (1975)). Alternatively, anthracyclines have been linked toantibodies through carbodiimide-mediated linkage of the amino group ofthe anthracycline to carboxyl groups on the antibody (Hurwitz et al.,supra) or an aminoalkyl group (Hurwitz et al., "The Effect in vivo ofChemotherapeutic drug-antibody conjugates in two murine experimentaltumor systems" Int. J. Cancer 21:747-755 (1978)). These linkages are noteasily hydrolyzed and make it difficult to control the release of theanthracycline. Anthracyclines have also been linked to antibodies bycross-linking the amino sugar of the drug and amino groups on theantibody with glutaraldehyde (Belles-Isles et al., "In Vitro Activity ofDaunomycin-Anti-AlphaFetoprotein Conjugate On Mouse Hepatoma Cells", Br.J. Cancer 41, pp. 841-42 (1980)). However, studies with immunoconjugatesin which the amino sugar portion of the anthracycline molecule wasmodified by linkage to the antibody indicate a loss of cytotoxicactivity of the conjugated drug (Arnon et al., supra, at pp. 7-8). Inaddition, studies of anthracycline analogs indicate that modificationsof anthracyclines at their amino sugars result in a decrease in thecytotoxic activity of the drug analog relative to the parent drug(Yamamoto et al., "Antitumor Activity of Some Derivatives of DaunomycinAt The Amino And Methyl Ketone Functions", J. Med. Chem. 15:872-75(1972)).

Still other immunoconjugates have been prepared wherein theanthracycline DAU has been linked directly to an antibody at thecarbon-14 (C-14) position of the drug. However, the selective cytotoxicactivity of these immunoconjugates toward tumor cells was not easilyreproducible and was revealed consistently only at a concentration of 20μg/ml (Gallego et al., supra).

Japanese patent application 274658 discloses the conjugation of ananthracycline to an antibody via a C-13 acylhydrazone linkage. Thisconjugation was accomplished using methods that involve derivatizationof the antibody and subsequent reaction of that derivative withanthracycline. These methods are not favored because derivatization ofthe antibody involves undesirable non-specific reactions and yields verylow anthracycline:antibody ratios. According to the first method, theantibody was treated with carbodiimide in the presence of hydrazine toyield a hydrazide antibody derivative which was then reacted with theanthracycline such that the anthracycline was linked directly to theantibody structure. The resulting immunoconjugates, however, are proneto aggregation of the antibody molecules. Furthermore, because thismethod requires carboxylic groups which may be limited in number, theseimmunoconjugates have low anthracycline:antibody ratios (approximately1.1-1.3). The second method involves reacting the antibody with succinicanhydride to yield a hemi-succinate derivative of the antibody. Thisderivative was next reacted with hydrazine to yield an antibodyhydrazide derivative which was then reacted with the anthracycline,daunomycin. This second approach is flawed in that the reaction of theantibody derivative with hydrazine is non-specific, leading to theproduction of a mixture of different antibody derivatives in addition tothe desired hydrazide derivative. Thus, as indicated in the 274658application, the molar ratio of anthracycline to antibody was very low(approximately 1, see Japanese application, page 264, column 1). Seealso, European patent application, Publication No. 294294, whichdiscloses the conjugation of a C-13 hydrazone derivative of ananthracycline to the carbohydrate moiety of an antibody.

Other anthracycline hydrazones are disclosed in Tong et al., J. Med.Chem., 21:732-37 (1978); Smith et al., J. Med. Chem.. 21:280-83 (1978);and Brownlee et al., J. Chem. Soc., pp. 659-61 (1986). See also U.S.Pat. No. 4,112,217, which discloses bis-hydrazones of DAU and ADM. Inother studies, anthracyclines have been linked to high molecular weightcarriers, such as dextran or polyglutamic acid, in order to potentiatethe cytotoxic activity and reduce the toxicity of the drug (Arnon etal., supra, at p. 5 and Hurwitz et al., "Soluble Macromolecules AsCarriers For Daunorubicin", J. Appl. Biochem. 2, pp. 25-35 (1980)).These carrier-linked anthracyclines have also been covalently bound toantibodies directed against tumor-associated antigens to formimmunoconjugates for targeting of the cytotoxic drug specifically totumor cells. For example, ADM has been linked to such an "anti-tumor"antibody via a carboxy-methyl-dextran hydrazide bridge wherein the ADMmolecule was linked to a hydrazine derivative of carboxymethyl dextranat the C-13 carbonyl of the ADM to form a hydrazone. The antibody wasthen linked to the dextran hydrazide derivative with glutaraldehyde toform an adriamycin-dexantibody conjugate (Arnon et al., "MonoclonalAntibodies As Carriers For Immunotargeting Of Drugs", in MonoclonalAntibodies For Cancer Detection And Therapy, Baldwin et al. (Eds.), pp.365-83 (1985) and Hurwitz et al., "A Conjugate 0f Adriamycin AndMonoclonal Antibodies To Thy-1 Antigen Inhibits A Human NeuroblastomaCells In Vitro", Ann. N.Y. Acad. Sci. 417, pp. 125-36 (1983)).

However, the use of carriers entails certain disadvantages. For example,carrier-containing immunoconjugates are quite large in size and areremoved rapidly by the reticuloendothelial system in vivo (Dillman etal., "Preclinical Trials With Combinations And Conjugates Of T101Monoclonal Antibody And Doxorubicin", Cancer Res. 46:4886-91 (1986)).This rapid removal of the carrier-containing immunoconjugates may not beadvantageous for therapy because the conjugated drug may never reach itsintended site of action, i.e., the target group of cells to be killed.In addition, the presence of the high molecular weight carrier maynegatively affect the stability of the immunoconjugate and has beenshown to reduce the binding activity of the antibody of the conjugate(Embleton et al., "Antibody Targeting Of Anti-Cancer Agents", inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(Eds.), pp. 323-24 (1985)). Furthermore, in studies with tumor cells,there is no evidence that high molecular weight carrier-containingimmunoconjugates are able to localize to the tumor cells in vivo.(Compare Ford et al., "Localization And Toxicity Study Of AVindesine-Anti-CEA Conjugate In Patients With Advanced Cancer" , Br. J.Cancer 47:35-42 (1983), which demonstrates localization ofdirectly-conjugated drug-antibody conjugates to tumor cells in vivo).

Thus, the conjugation of anthracyclines to antibodies by the use ofspecific linkages and carriers has been disclosed. As outlined above,the use of these immunoconjugates entails distinct disadvantagesdepending upon the specific linkage or carrier used.

Certain ligand-toxin conjugates have also been disclosed. U.S. Pat. No.4,545,985, issued to Pastan, discloses an exotoxin conjugate whereinPseudomonas exotoxin (PE) is linked to EGF in a ratio of 1:2 for useagainst cells having large numbers of EGF receptors. EGF-ricin A andEGF-diphtheria toxin conjugates have also been made; Cawley et al.,"Epidermal Growth Factor-Toxin A Chain Conjugates:" EGF-Ricin A Is APotent Toxin While EGF-Diphtheria Fragment A Is Nontoxic", Cell22:563-70 (1980) and Shimizu et al., "A Cytotoxic Epidermal GrowthFactor Cross-Linked To Diphtheria Toxin A-Fragment", FEBS Letters 118(No.2):274-78 (1980)). Furthermore, Pseudomonas exotoxin fusion proteinshave been prepared using proteins, polypeptides and growth factors suchas TGF-α, IL-2, IL-6 and CD4 (Pastan et al., "Novel Cytotoxic AgentsCreated By The Fusion Of Growth Factor And Toxin Genes", FourthInternatl. Conference On Monoclonal Antibody Immunoconjugates ForCancer, p. 36 (Mar. 30-Apr. 1, 1989); Lorberboum et al., Proc. Natl.Acad. Sci. USA, 85:1922-26 (1988); Chaudhary et al., Proc. Natl. Acad.Sci. USA, 84:4538-42 (1987); Siegall et al., Proc. Natl. Acad. Sci. USA,85:9738-42 (1988); and Chaudhary et al., Nature, 335:369-72 (1988)). Adiphtheria toxin-α-melanocyte stimulating hormone fusion protein hasbeen made (Murphy et al., "Genetic Construction, Expression AndMelanoma-Selective Cytotoxicity Of A Diphtheria Toxin-Relatedα-Melanocyte-Stimulating Hormone Fusion Protein", Proc. Natl. Acad. Sci.USA, 83:8258-62 (1986), and U.S. Pat. No. 4,675,382, issued to Murphy).Ligand conjugates comprising protein toxins, however, may prove to beimmunogenic in xenogeneic hosts.

In addition, anthracyclines such as ADM or DAU have been chemicallylinked to certain protein or polypeptide ligands such as transferrin(United Kingdom patent application, GB 2116979 A) and melanotropin(Varga et al., "Melanotropin-Daunomycin Conjugate ShowsReceptor-Mediated Cytotoxicity For Cultured Murine Melanoma Cells",Nature 267: 56-58 (1977)). PCT patent application WO 88/00837 describesEGF linked via a polymeric carrier to a cytotoxic substance such as DAUand U.S. Pat. Nos. 4,522,750 and 4,590,001 describe transferrin linkedto vinca alkaloid and platinum, respectively.

The cytotoxic drug to be used in the immunoconjugate should be releasedvia a conditional release mechanism, i.e. the cytotoxic drug should bereleased at a specific site rather than by a gradual, nonspecific sitehydrolysis. It has been proposed that particular immunoconjugates aretranslocated to lysosomes (deDuve, "Lysosomes Revisited", Eur. J.Biochem. 137:391-397 (1983)), which are slightly acidic (pH 5.0 to 5.5)(Poznansky and Juliano, "Biological Approaches to the ControlledDelivery of Drugs: A Critical Review", Pharmacol. Rev. 36:277-336(1984)). The use of acidic conditions to release conjugated drug hasbeen reported in the development of cis-aconityl linkers to ADM (Shenand Reiser, "Cis-Aconityl Spacer Between Daunomycin and MacromolecularCarriers: A Model of pH-sensitive Linkage Releasing Drug From aLysosomotrophic Conjugate", Biochem. Biophys. Res. Commun. 102:1048-1054(1981) and Yang and Reisfeld, "Doxorubicin Conjugates With a MonoclonalAntibody Directed to a Human Melanoma-Associated Proteoglycan Suppressesthe Growth of Established Tumor Xenografts in Nude Mice", Proc. Natl.Acad. Sci. 85:1189-1193 (1988)), and ketal linkers to diphtheria toxin(Srinivasachar and Neville, "New Protein Cross-Linking Reagents That AreCleaved by Mild Acid", Biochemistry 28:2501-2509 (1989)).

Greenfield et al. have recently described the formation ofacid-sensitive immunoconjugates containing the acylhydrazine compound,3-(2-pyridyldithio)proprionyl hydrazide conjugated via an acylhydrazonebond to the 13-keto position of the anthracycline molecule, andconjugation of this anthracycline derivative to an antibody or ligandmolecule (Greenfield et al., European Patent Application No. 328,147,published Aug. 16, 1989).

It would be useful to provide additional bifunctional compounds that arestructured to provide acid-sensitive linkage between molecules,including targeting and reagent molecules for use in therapy in vivo.

SUMMARY OF THE INVENTION

The present invention provides novel bifunctional compounds that arereadily conjugated with useful molecules, and methods for preparing thebifunctional compounds. The bifunctional compounds contain a reactivepyridinyldithio or ortho-nitrophenyldithio group. The invention alsoprovides novel conjugates containing cytotoxic molecules linked to thebifunctional compounds to form derivatives of the cytotoxic moleculesand further conjugates containing the cytotoxic derivatives linked to amolecule capable of reacting with a target cell population to be killed.This targeting molecule can be a protein such as an antibody or a ligandsuch as bombesin or EGF.

According to one embodiment, a novel bifunctional compound,N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide (compound 10) issynthesized and is used to form a semicarbazone derivative of ADMcontaining a semicarbazone bond at the C-13 position of the ADM thatserves as the site of attachment of the ADM to compound 10.

According to another preferred embodiment, a novel bifunctionalcompound, 2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]carbonicdihydrazide (compound 11a) is synthesized and is used to form acarbazone derivative of ADM containing a carbazone bond at the C-13position of the ADM that serves as the site of attachment of the ADM tocompound 11a.

In another preferred embodiment, a novel bifunctional compound,N-[4-[(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide (compound12) is synthesized and is used to form a thiosemicarbazone derivative ofADM containing a thiosemicarbazone bond at the C-13 position of the ADMthat serves as the site of attachment of the ADM to compound 12.

According to another preferred embodiment, a novel bifunctionalcompound, 2[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate (compound 13)is synthesized and is used to form a hydrazone derivative of ADMcontaining a carboxylatehydrazone bond at the C-13 position of the ADMthat serves as the site of attachment of the ADM to compound 13.

In yet another preferred embodiment a novel bifunctional compound,N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinobenzamide (compound 15), issynthesized and is used to form an arylhydrazone derivative of ADMcontaining an arylhydrazone bond at the C-13 position of the ADM thatserves as the site of attachment of the ADM to compound 15.

According to still another embodiment of this invention, a number ofmolecules of the above novel anthracycline derivatives are linked to amolecule reactive with a selected target cell population. Preferably,the cell-reactive-molecule is an antibody, and is a monoclonal antibody.Each anthracycline derivative molecule is linked to the antibody via thebifunctional compound bound to the anthracycline via a semicarbazone,carbazone, thiosemicarbazone, carboxylatehydrazone or arylhydrazone bondat the C-13 position of the anthracycline molecule to form the novelimmunoconjugates of the invention. For example, a preferred embodimentof the invention involves the synthesis of a novel adriamycin derivativemolecule that is condensed with a thiolated antibody resulting in theattachment of the anthracycline to the antibody via the bifunctionalcompound. The hydrazone bond formed at the C-13 position of the ADMserves as the site of attachment to the ADM. In this embodiment adisulfide bond is present within the bifunctional compound through whichit is attached to the antibody. According to another preferredembodiment, the adriamycin derivative molecule (ADM bound to thebifunctional compound) is reduced to generate a sulfhydryl group and theresulting derivative is condensed with a maleimide-derivatized antibody.This leads to the formation of an immunoconjugate having a N-substitutedhydrazone bond as the site of the bifunctional compound attachment tothe C-13 position of ADM and a thioether bond within the bifunctionalcompound through which it is attached to the antibody.

According to yet another preferred embodiment of the invention, thenovel anthracycline derivatives may be covalently linked to ligands,such as bombesin, transferrin or EGF, resulting in the attachment of theanthracycline to the ligand via a bifunctional compound. As in the otherembodiments described above, the anthracycline is attached to thebifunctional compound via a hydrazone bond formed at the C-13 positionof the anthracycline. The ligand is preferably thiolated prior tolinkage to the anthracycline derivative, but it may also be directlyattached to ligands having an endogenous free thiol group.

As is evident from these embodiments, the present invention providesnovel bifunctional compounds and derivatives of anthracyclines useful inthe preparation of the conjugates of this invention.

The immunoconjugates of the present invention have an anthracycline:antibody molar ratio of at least 1:1 and up to 10:1, and preferably ofapproximately 4:1 to 10:1, and retain both antibody and cytotoxic drugactivity for the killing of selected target cells. Theanthracycline-ligand conjugates described herein preferably have ananthracycline:ligand ratio of at least 1:1 and up to 10:1, andpreferably of approximately 4:1 to 10:1. The acid-sensitive bond that ispresent at the site of attachment of the anthracycline to thebifunctional compound of these conjugates is ideally suited for therelease of active drug under acidic conditions such as those typicallyencountered within a cell, e.g., in lysosomal vesicles.

The release of adriamycin by hydrolysis of each of the above namedderivatives, as a function of pH, demonstrated that the new derivativeshad wide ranging release rates under acidic conditions mimicking thelysosomal environment. These derivatives also demonstrated cytotoxicityas immunoconjugates with the anti-transferrin receptor monoclonalantibody 5E9.

The N-substituted hydrazine bifunctional compounds contain a hydrazinemoiety, and a reactive pyridinyldithio or ortho-nitrophenyldithiomoiety. These novel bifunctional compounds may be used to link a varietyof molecules to form useful conjugates. The molecule to be linked to thehydrazine moiety of the bifunctional compound contains a free carbonylgroup, or a group that is derivatized to contain a carbonyl group, suchas a cytotoxic reagent molecule. When the molecule containing thecarbonyl group is linked to the hydrazine moiety of the bifunctionalcompound a hydrazone bond is formed which is a semicarbazone, carbazone,thiosemicarbazone, carboxylatehydrazone or arylhydrazone bond, dependingon which bifunctional compound of the invention is used to form theconjugate. The molecule to be linked to the end of the bifunctionalcompound that contains the pyridinyldithio or ortho-nitrophenyldithiomoiety contains a free sulfhydryl group or a group that can bederivatized to contain a sulfhydryl group, such as an antibody moleculeor ligand that is preferably reactive with antigens or receptors on thetarget cells to be killed. The pyridinyldithio moiety leaves during thereaction of the antibody with the bifunctional compound. The moleculecontaining a free carbonyl group is preferably a cytotoxic reagentmolecule such as an anthracycline capable of killing selected cells. Ina preferred embodiment, the hydrazone bond linking the cytotoxic reagentmolecule to the bifunctional compound permits pH sensitive release ofthe cytotoxic reagent.

The conjugates of this invention formed by linking molecules with thebifunctional compounds of the invention may be used in pharmaceuticalcompositions, such as those comprising a pharmaceutically effectiveamount of at least one immunoconjugate of the invention and apharmaceutically acceptable carrier. The present invention alsoencompasses methods for the selective delivery of cytotoxic reagents toa selected population of target cells desired to be eliminated, as wellas methods for treating a mammal in a pharmaceutically acceptable mannerwith a pharmaceutically effective amount of the compositions of theinvention.

Advantageously, the compounds, conjugates, pharmaceutical compositions,and methods disclosed herein provide a useful approach to the targetingof cytotoxic reagents to a selected population of cells for thepreferential killing of those target cells in the treatment of diseasessuch as cancers and other tumors, non-cytocidal viral or otherpathogenic infections, and autoimmune disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structures of the novel adriamycin derivatives of theinvention formed by reacting the bifunctional compounds of the inventionwith adriamycin as described in Examples 1-5, infra.

FIG. 2 depicts in schematic form the synthesis of theN-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide bifunctionalcompound used to prepare the semicarbazone derivative of adriamycin asdescribed in Example 1, infra.

FIG. 3 depicts in schematic form the synthesis of the2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]carbonic dihydrazidebifunctional compound used to prepare the carbazone derivative ofadriamycin as described in Example 2, infra.

FIG. 4 depicts in schematic form the synthesis of theN-[4-[(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamidebifunctional compound used to prepare the thiosemicarbazone derivativeof adriamycin as described in Example 3, infra.

FIG. 5 depicts in schematic form the synthesis of the2[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate bifunctional compoundused to prepare the carboxylatehydrazone derivative of adriamycin asdescribed in Example 4, infra.

FIG. 6 depicts in schematic form the synthesis of theN-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide bifunctionalcompound used to prepare the arylhydrazone derivative of adriamycin asdescribed in Example 5, infra.

FIG. 7 depicts in schematic form the preparation of immunoconjugates ofthe invention using an antibody thiolated with SPDP reacted with thebifunctional compounds of the invention, as described in Example 7,infra.

FIG. 8 depicts in schematic form the preparation of immunoconjugates ofthe invention using an antibody thiolated with 2-IT reacted with thebifunctional compounds of the invention.

FIG. 9 depicts in schematic form the preparation of immunoconjugates ofthe invention having a thioether linkage between the antibody and thereduced bifunctional compound using a antibody reacted with SMPB to addmaleimide groups.

FIG. 10 is a graph of the release of adriamycin as a function of timeafter incubation of the adriamycin derivatives of the invention inbuffer at pH 4.5, as described in Example 6, infra.

FIG. 11 is a graph of the release of adriamycin as a function of timeafter incubation of the adriamycin derivatives of the invention inbuffer at pH 5.0, as described in Example 6, infra.

FIG. 12 is a graph of the release of adriamycin as a function of timeafter incubation of the adriamycin derivatives of the invention inbuffer at pH 7.4, as described in Example 6, infra.

FIG. 13 is a graph of the release of adriamycin from the carbazonederivative of adriamycin and from a 5E9 immunoconjugate of thisderivative, as a function of time after incubation in buffer at pH 4.5,as described in Example 7, infra.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein disclosed may be more fullyunderstood, the following detailed description is set forth.

The present invention relates to novel N-substituted hydrazinebifunctional compounds:N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide (compound 10);2-[[[2-[(2-pyridinyl)dithio]- ethyl]amino]carbonyl]carbonic dihydrazide(compound 11a);N-[4-[(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide (compound12); 2-[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate (compound 13);and N-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide (compound 15).These compounds are used to form novel N-substituted hydrazonederivatives of cytotoxic reagents such as anthracyclines, and whenjoined to an antibody, to form immunoconjugates. The invention alsorelates to the methods for production of the bifunctional compounds,cytotoxic derivatives and immunoconjugates, and to pharmaceuticalcompositions and methods for delivering cytotoxic reagents to targetcells to treat diseases such as cancers and other tumors, non-cytocidalviral or other pathogenic infections and autoimmune disorders.

The conjugates comprise at least one cytotoxic derivative moleculeconnected by one of the bifunctional compounds to at least one moleculethat is reactive with the target cell population. This molecule can be aprotein such as an antibody, preferably a monoclonal antibody, or aligand such as bombesin or EGF.

Thus, according to one preferred embodiment, the novel compound,N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide, compound 12, wassynthesized and used to form the semicarbazone derivative of ADMcontaining a semicarbazone bond at the C-13 position of the ADM. Inanother preferred embodiment, the novel compound2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]carbonic dihydrazide,compound 11a, was synthesized and used to form the carbazone derivativeof ADM having a carbazone bond at the C-13 position of the ADM.According to another preferred embodiment, the novel compoundN-[4-[(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide, compound12, was synthesized and used to form the thiosemicarbazone derivative ofADM having a thiosemicarbazone bond at the C-13 position of ADM. In yetanother preferred embodiment the novel compound2[(2-pyridinyl)dithio]ethylhydrazinecarboxylate, compound 13, wassynthesized and used to form the hydrazone derivative of ADM having acarboxylatehydrazone bond at the C-13 position of the ADM. In stillanother preferred embodiment, the novel compound, N-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide, compound 15, was usedto form the arylhydrazone derivative of ADM having an arylhydrazone bondat the C-13 position of ADM.

In other embodiments the invention relates to conjugates containing atleast one molecule reactive with a target cell population, such as anantibody or ligand, and at least one cytotoxic molecule that killscells, linked by the novel bifunctional compounds of the invention.Thus, according to another preferred embodiment, the invention relatesto immunoconjugates containing an antibody directed against a targetcell population, for example a tumor cell population, the antibodyhaving a number of anthracycline derivative molecules linked to itsstructure. The anthracycline derivative molecules are attached to athiolated antibody covalently such that a disulfide bond is formedbetween each drug molecule and an antibody, the bifunctional compoundbeing attached to the anthracycline derivative by a hydrazone bond atthe C-13 position of the anthracycline. More than one drug molecule maybe attached to each antibody molecule using one bifunctional compound ofthe invention per drug molecule. A molar ratio of 4:1 indicates that 4drug (i.e. anthracycline derivative) molecules are attached to a singleantibody.

In an alternative embodiment, the anthracycline derivative is reduced tocreate a sulfhydryl group and this ADM derivative is condensed with amaleimide-modified antibody forming a thioether bond between theantibody and the anthracycline.

These conjugates permit the pH-sensitive release of unmodifiedanthracycline drug to prevent structural modifications of the drug thatmight result in reduction of cytotoxicity.

In yet another preferred embodiment, the invention encompassesanthracycline-ligand conjugates comprised of a ligand, such as apolypeptide or peptide ligand, that reacts with one or more receptorsassociated with the cell surface of a target cell population, the ligandhaving at least one anthracycline derivative molecule linked to itsstructure. The anthracycline is covalently bound to the peptide by abifunctional compound that is attached to the anthracycline at the C-13position of the anthracycline via a hydrazone bond. In an alternativeembodiment, the anthracycline derivative is reduced to create asulfhydryl group and this derivative is then condensed with amaleimide-modified ligand.

The conjugates of this invention can be prepared in a stepwise fashionby the initial formation of a novel N-substituted hydrazine compoundthat is used to form a hydrazone derivative of the cytotoxic reagentwhich is then reacted with a protein or ligand of the appropriatespecificity (see Hardy, "Purification And Coupling Of FluorescentProteins For Use In Flow Cytometry", in Handbook Of ExperimentalImmunology, Volume 1: Immunochemistry, D. M. Weir et al. (Eds.), pp.31.4-31.12 (4th Ed. 1986) for a discussion of conventional antibodycoupling techniques and Varga et al., supra, for the preparation ofligand conjugates).

The length of the bifunctional compound that connects the cytotoxicreagent with the cell-reactive component of the conjugates may vary aslong as the bifunctional compound is attached via one of theaforementioned hydrazone bonds to the carbonyl group of the cytotoxicreagent molecule or molecules.

The cytotoxic reagents that comprise the conjugates of this inventionmay be any molecule containing a carbonyl group. Such reagents include,but are not limited to, the anthracyclines: adriamycin, daunomycin,detorubicin, carminomycin, idarubicin, epirubicin, esorubicin,4'-THP-adriamycin, AD-32, and3'-deamino-3'-(3-cyano-4-morpholinyl)doxurubicin (Casazza, "ExperimentalStudies On New Anthracyclines", in Adriamycin: Its Expanding Role InCancer Treatment, M. Ogawa et al. (Eds.), pp. 439-52 (Excerpta Medica,1984)).

It is to be understood that the cell-reactive molecule to which thecytotoxic reagent is linked in the conjugate via the bifunctionalcompound can be any molecule that binds to or reacts with the cellpopulation sought to be eliminated and which possesses a sulfhydrylgroup or can be modified to contain a sulfhydryl or maleimide group.Such molecules include, but are not limited to, large molecular weightproteins (generally, greater than 10,000 daltons) such as antibodies,smaller molecular weight proteins (generally, less than 10,000 daltons),polypeptide or peptide ligands, and non-peptidyl ligands.

Antibodies that comprise the immunoconjugates of this invention may beany antibody reactive with a specific target cell population desired tobe eliminated or killed. Examples of such antibodies include, but arenot limited to, antibodies that bind to tumor-associated antigens suchas antigens found on carcinomas, melanomas, lymphomas, bone or softtissue sarcomas, as well as other tumors, antibodies that bind to virus-or other pathogen-associated antigens, and antibodies that bind toabnormal cell surface antigens. These antibodies may be polyclonal orpreferably, monoclonal, and can be produced using techniques wellestablished in the art (DeWeger et al., Eradication Of Murine LymphomaAnd Melanoma Cells By Chlorambucil-Antibody Complexes", ImmunologicalRev. 62:29-45 (1982) (tumor-specific polyclonal antibodies produced andused in conjugates); Yeh et al., "Cell Surface Antigens Of HumanMelanoma Identified By Monoclonal Antibody," Proc. Natl. Acad. Sci.76:2927-31 (1979), and Brown et al., "Structural Characterization OfHuman Melanoma-Associated Antigen p97 With Monoclonal Antibodies," J.Immunol. 127 (No.2):539-46 (1981) (tumor-specific monoclonal antibodiesproduced)). For example, the monoclonal antibody, L6, specific for humanlung carcinoma cells or the monoclonal antibody, 791T/36, specific forosteogenic sarcoma cells, can be used. Furthermore, noninternalizing orpreferably, internalizing antibodies may be used. The term "antibody" asused in this application includes intact antibody molecules or fragmentscontaining the active binding region of the antibody molecule, e.g., Fabor F(ab')₂. If monoclonal antibodies are used, the antibodies may be of,but are not limited to, mouse or human origin, or chimeric antibodies.

It is also to be understood the term "ligand" as used herein includesany molecule that binds specifically to a receptor associated with thecell surface of a target cell population. Preferred ligands that can beused to form the anthracycline-ligand conjugates of this inventioninclude, but are not limited to, protein, polypeptide, or peptideligands such as transferrin, epidermal growth factor (EGF), bombesin,gastrin, gastrin-releasing peptide, platelet-derived growth factor,IL-2, IL-6, tumor growth factors (TGF)-α and TGF-β, vaccinia growthfactor (VGF), insulin and insulin-like growth factors I and II. Othernon-peptidyl ligands include steroids, carbohydrates and lectins.

Thus, the cell-reactive "targeting" molecule, e.g., antibody or ligand,of the conjugates of this invention acts to deliver the cytotoxicreagent molecules to the particular target cell population with whichthe antibody or ligand is reactive. For example, an antibody directedagainst an antigen found on the surface of tumor cells will bind to anddeliver the cytotoxic reagents to those tumor cells or an antibodydirected against a protein of the Human Immunodeficiency Virus (HIV)that causes AIDS will deliver its cytotoxic reagents to HIV-infectedcells. Similarly, because tumor cells, such as carcinomas,preferentially express certain receptors at high density, such as theEGF receptor, a ligand such as EGF will bind to and deliver thecytotoxic reagent to carcinoma cells.

Release of the cytotoxic reagent within or at the site of the particularcell population with which the antibody or ligand reacts results in thepreferential killing of those particular cells. Thus, it is apparentthat the conjugates of this invention are useful in the treatment of anydisease wherein a specific cell population is sought to be eliminated,the cell population having a cell surface antigen or receptor whichallows binding of the conjugate. Diseases for which the presentconjugates are useful include, but are not limited to, cancers and othertumors, non-cytocidal viral or other pathogenic infections such as AIDS,herpes, CMV (cytomegalovirus), EBV (Epstein Barr Virus), and SSPE(subacute schlerosis panencephalitis), and rheumatoid arthritis.

Without being bound by theory, it is believed that the antibody- orligand-linked cytotoxic reagent molecules, i.e., in the form of theconjugates of the invention, are delivered to the target cells to bekilled via the antibody or ligand specificity and may then enter thecell via the same endocytic pathway that leads to internalization ofmembrane-bound unconjugated antibodies and ligands (Pastan et al.,"Pathway Of Endocytosis", in Endocytosis, I. Pastan et al. (Eds.), pp.1-44 (Plenum Press, 1985)). Once inside the cell, the endocytic vesiclescontaining the conjugate fuse with primary lysosomes to form secondarylysosomes (Embleton et al., supra, at p. 334). Because the cytotoxicmolecules are bound to the antibody or ligand component of the conjugatevia acid-sensitive hydrazone bonds, exposure of the conjugate to theacid environment of the endocytic vesicles and lysosomes results in therelease of the cytotoxic reagent from the conjugate. Furthermore, thereagent released is believed to be in the form of a relativelyunmodified reagent capable of full cytotoxic activity. Thus, theacid-sensitive bond of the conjugate is highly advantageous for therelease of the cytotoxic reagent within target cells, enhancing thecytotoxicity of the conjugate toward those cells. Alternatively, thehydrazone bond may be cleaved under acidic and reducing conditions inthe immediate environment external to or surrounding the target cells,e.g., at the site of a tumor, and the released drug may be taken up bythe tumor cells.

The novel bifunctional compounds, derivatives of cytotoxic reagents andconjugates of the invention, and methods for their production, areexemplified by preferred embodiments in which the anthracycline,adriamycin, was used. In general, carbonyl derivatives of adriamycinwere prepared by treating adriamycin hydrochloride with one of the fivebifunctional compounds of the invention in methanol at room temperature.It was found that addition of catalytic amounts of trifluoroacetic acid(TFA) accelerated the condensation reactions so that the reaction wascomplete after an overnight stirring. Few side-products were produced inthese reactions and the purification procedure only requiredprecipitation with acetonitrile. These simplified procedures representan improvement over those previously reported by Greenfield et al.,supra. in that they are easier to perform, more economical, and faster,and provide additional, novel bifunctional compounds for conjugating avariety of molecules.

In a first embodiment, a novel bifunctional compound was prepared byfirst reacting methoxycarbonylsulfenyl chloride with 2-aminoethanethiolhydrochloride, then 2-mercaptopyridine to form2-[(2-pyridinyl)dithio]ethanamine hydrochloride (see FIG. 2). Thiscompound was then reacted with phosgene in the presence of triethylamine(TEA), then t-butyl carbazate, to formN-[2-[(2-pyridinyl)dithio]ethyl]-2-(tert.-butoxy-carbonyl)hydrazinecarboxamide.The hydrazinecarboxamide was next dissolved in TFA to formN-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide, compound 10, asemicarbazide, which was then reacted with adriamycin hydrochloride toform the semicarbazone derivative of ADM containing a reactivepyridinyldithio moiety, compound 1, FIG. 1.

In another preferred embodiment, a novel carbazide bifunctional compoundwas prepared (FIG. 3). The compound t-butyl carbazate was reacted withtriphosgene in the presence of TEA. 2-(2-pyridinyldithio)ethanaminehydrochloride was then added to form2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]-2,2'-bis(tert.-butoxycarbonyl)carbonicdihydrazide. This intermediate was added to TFA to form2-[[[2-[(2-pyridinyl)- dithio]ethyl]amino]carbonyl]carbonic dihydrazide,compound 11a, which was then added to adriamycin hydrochloride to formthe carbazone derivative of ADM (compound 2, FIG. 1) containing areactive pyridinyldithio moiety.

In yet another preferred embodiment, a novel thiosemicarbazidebifunctional compound was formed (FIG. 4). In this embodiment, potassiumphthalimide was reacted with 1,4-dibromo-2-butene to form1-bromo-4-(N-phtalimido)-2-butene. This compound was reacted withpotassium thioacetate to form 1-(acetylthio)-4-(N-phthalimido)-2-butenewhich was then reacted with hydrazine and was treated withmethoxycarbonylsulfenyl chloride followed by 2-mercaptopyridine to form1-amino-4-[(2-pyridinyl)dithio]-2-butene hydrochloride. This compoundwas combined with TEA followed by di-2-pyridyl thionocarbonate, thent-butyl carbazate was added to form the t-boc derivative ofN-[4-[(2-pyridinyl)dithio]-2-butenyl]-hydrazinecarbothioamide, compound12. This compound was dissolved in TFA to form a compound in the form ofa gum, which was then reacted with adriamycin hydrochloride and TFA toform the thiosemicarbazone derivative of ADM having a thiosemicarbazonebond at the C-13 position of the ADM (compound 3, FIG. 1) and having areactive pyridinyldithio moiety.

Another preferred embodiment involves the formation of yet another novelbifunctional compound (FIG. 5). Chlorocarbonyl sulfenyl chloride wasreacted with 2mercaptoethanol and 2-mercaptopyridine. Ammonium carbonatesolution was added to form 2-(2-Pyridinyl)dithio)ethanol as a colorlessoil. Carbonyldiimidazole was added and the mixture was reacted withhydrazine to form 2-[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate,compound 13. This compound was reacted with adriamycin hydrochloride andTFA and then acetonitrile to form the carboxylatehydrazone derivative ofADM (compound 4, FIG. 1) having a carboxylatehydrazone bond at the C-13position of the ADM and having a reactive pyridinyldithio moiety.

In still another preferred embodiment a novel bifunctional compound wassynthesized (FIG. 6). This compound was prepared by reactingp-hydrazinobenzoic acid with di-t-butylpyrocarbonate to form4-(N-boc-hydrazino)benzoic acid. This compound was reacted withN-hydroxysuccinimide and DCC to give the N-hydroxysuccinimide ester of4-N-boc(hydrazino) benzoic acid. This material was reacted with2-(2-pyridinyl)dithio)ethanamine hydrochloride and TEA to formN-[2-(2-pyridinyl)dithio]ethyl-4-N-boc-(hydrazino)benzamide. Thiscompound was then treated with TFA to formN-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide, compound 15,which was then reacted with adriamycin hydrochloride to form thearylhydrazone derivative of ADM (compound 5, FIG. 1) having anarylhydrazone bond at the C-13 position of ADM and having a reactivepyridinyldithio moiety.

The novel N-substituted hydrazone derivatives of ADM described abovewere used to form the conjugates of the invention. Each derivative wasreacted with a monoclonal antibody that had been previously thiolatedwith SPDP or with 2-IT (2-iminothiolane) as shown in FIGS. 7 and 8,respectively. The resulting immunoconjugates were comprised of ADMmolecules conjugated to the monoclonal antibody by means of thebifunctional compound attached to the C-13 position of each ADM moleculethrough a hydrazone bond. The bifunctional compounds also contained adisulfide bond through which each was attached to the antibody.

The bifunctional compound connecting the ADM and the antibody may becomprised of a number of constituents and linkages as long as theselinkages include an acid-sensitive hydrazone bond at the C-13 positionof the anthracycline. The antibody of the preferred embodiments wasmonoclonal antibody 5E9.

In another embodiment of the invention, the novel bifunctional compoundsare combined with adriamycin to form a derivative which is then furthertreated with the reducing agent dithiotreitol (DTT) ortributylphosphine, to produce an adriamycin derivative containing asulfhydryl (-SH) group at the end of the bifunctional compound. Thisderivative is then reacted with a monoclonal antibody or ligand to whichmaleimide groups have been attached, for example, by reaction of theantibody with succinimidyl-4-(p-maleimidophenyl) butyrate (SMPB). Animmunoconjugate is formed that has a bifunctional compound attached by ahydrazone bond at the C-13 position of each ADM and has a thioetherlinkage as part of the attachment to the antibody (see FIG. 9).

Thus, it is apparent that the bifunctional compound connecting the ADMand antibody or ligand may be comprised of a number of constituents andlinkages as long as these linkages include a hydrazone bond at the13-keto position of the ADM and a reactive pyridinyldithio ororthonitrophenyldithio group for connecting to the antibody.

According to another embodiment, the novel ADM derivatives of theinvention are reacted with a ligand such as bombesin, EGF ortransferrin, the ligand having first been derivatized to possess thiolgroups or maleimide groups. In the case of bombesin, a cysteine residueis introduced onto the amino terminus of the peptide to provide areactive sulfhydryl group for conjugation with the ADM derivative. Inthe case of murine EGF, the polypeptide is reacted with SPDP tointroduce a reactive sulfhydryl group at the amino terminus of themolecule for conjugation. In the case of transferrin, the protein isfirst reacted with 2-IT to introduce reactive thiol groups onto theprotein structure. In each case, the thiolated ligand is then reactedwith the ADM derivative to form an anthracycline-ligand conjugate of theinvention having a bifunctional compound between the ligand and the ADM,the bifunctional compound being attached to the C-13 position of eachanthracycline molecule via a hydrazone bond.

It is apparent that the present invention provides novel hydrazonederivatives of anthracyclines having the following general formula I:##STR1## wherein: R₁ is NHCONH(CH₂)_(n) SSR₈ ;

NHCONHNHCONH(CH₂)_(n) SSR₈ ;

NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) SSR₈ ;

NHCOO(CH₂)_(n) SSR₈ ;

NHArCONH(CH₂)_(n) SSR₈ ;

NCONH(CH₂)_(n) S--H;

NCONHNHCONH(CH₂)_(n) S--H;

NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) S--H;

NHCOO(CH₂)_(n) S--H; or

NHArCONH(CH₂)_(n) S--H; ##STR2## X is H, NO₂ or Halogen; ##STR3## m, nare integers from 1 to 10, which may be the same or different; R₂ isCH₃, CH₂ OH, CH₂ OCO(CH₂)₃ CH₃, or CH₂ OCOCH(OC₂ H₅)₂ ;

R₃ is OCH₃, OH or hydrogen;

R₄ is NH₂, NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl, 1-piperidinyl,4-methoxy-1-piperdinyl, benzyl amine, dibenzyl amine, cyanomethyl amineor 1-cyano-2-methoxyethyl amine;

R₅ is OH, 0-THP or hydrogen; and

R₆ is OH or hydrogen, provided that R₆ is not OH when R₅ is OH or O-THP.

and formula II: ##STR4## wherein: R₁ is NHCONH(CH₂)_(n) SSR₈ ;

NHCONHNHCONH(CH₂)_(n) SSR₈ ;

NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) SSR₈ ;

NHCOO(CH₂)_(n) SSR₈ ;

NH-Ar-CONH(CH₂)_(n) SSR₈ ;

NHCONH(CH₂)_(n) S--H;

NHCONHNHCONH(CH₂)_(n) S--H;

NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) S--H;

NHCOO(CH₂)_(n) S--H or

NH-Ar-CONH(CH₂)_(n) S--H ##STR5## X is H, NO₂ or halogen; ##STR6## m, nare integers from 1 to 10, which may be the same or different; R₂ isCH₃, CH₂ OH, CH₂ OCO(CH₂)₃ CH₃, or CH₂ OCOCH(OC₂ H₅)₂ ;

R₃ is OCH₃, OH or hydrogen;

R₄ and R₇ are independently hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl orsubstituted aralkyl; or R₄, R₇ and N together form a 4-7 membered ring,wherein said ring may be optionally substituted;

R₅ is OH, 0-THP or hydrogen; and

R₆ is OH or hydrogen, provided that R₆ is not OH when R₅ is OH or O-THP.

The above-disclosed bifunctional compounds and N-substituted hydrazonederivatives of anthracycline are novel compounds. The hydrazonederivatives of anthracycline may be used as novel cytotoxic reagents andalso represent intermediates in the preparation of the novel conjugatesof the invention. The hydrazone derivatives of anthracycline areexemplified by adriamycin semicarbazone; adriamycin carbazone;adriamycin thiosemicarbazone; adriamycin carboxylatehydrazone andadriamycin arylhydrazone, respectively, as described in the preferredembodiments discussed herein.

As can be seen from the above formulae, the N-substituted hydrazone ADMderivatives of the invention include N-substituted hydrazones of any ofa number of known anthracyclines such as adriamycin, daunomycin andcarminomycin. In addition, the derivatives include N-substitutedhydrazones derivatized at specific sites on the anthracycline structure(e.g., 4'-THP-adriamycin hydrazone and3'-deamino-3'-(3-cyano-4-morpholinyl)adriamycin hydrazone). These latterderivatives can be synthesized by first derivatizing the anthracyclineto form a desired analog and then using that analog to prepare theN-substituted hydrazone derivatives of the invention. Knownanthracycline analogs include those described in U.S. Pat. Nos.4,464,529 and 4,301,277 (3'-deamino-3'-(4-morpholinyl) or3'-deamino-3'(3-cyano-4-morpholinyl) anthracycline analogs), U.S. Pat.Nos. 4,202,967 and 4,314,054 (3'-deamino-3'-(1-piperdinyl)or3'-deamino-3'-(4-methoxy-1-piperdinyl) anthracycline analogs), U.S. Pat.No. 4,250,303 (N-benzyl or N,N-dibenzyl anthracycline analogs), U.S.Pat. No. 4,591,637 (N-methoxymethyl or N-cyanomethyl anthracyclineanalogs) and U.S. Pat. No. 4,303,785 (acetal analogs of anthracyclines).Thus, these known anthracycline analogs can be reacted as describedhereinabove to produce novel hydrazone derivatives of ADM which can thenbe conjugated to a cell-reactive molecule, such as an antibody or ligandof a desired specificity, as described above.

Alternatively, an underivatized N-substituted hydrazone derivative ofthis invention can first be produced as described herein from theunderivatized anthracycline, such as adriamycin, daunomycin orcarminomycin, and this novel derivative can then be derivatized toproduce a novel N-substituted hydrazone substituted as desired. Forexample, the semicarbazone ADM derivative can be derivatized at itsamino sugar moiety by reductive amination with 2,2'-oxydiacetaldehydeusing the procedure described in U.S. Pat. No. 4,464,529, to produce thesemicarbazene of '3-deamino-3'-(4-morpholino) anthracycline. Inaddition, the N-substituted hydrazone derivatives can be derivatized atthe R₅ position of formulae I and II, as described in U.S. Pat. No.4,303,785 to produce acetal derivatives of the hydrazone such as4'-THP-ADM N-substituted hydrazone.

It should be understood that these procedures for derivatizing thehydrazones of the invention can use as starting materials N-substitutedhydrazones of other reagents, including various chemotherapeutic agents.And, anthracyclines other than ADM, such as daunomycin or carminomycin,may be used to produce novel compounds such as [N-benzyl daunomycinN-substituted hydrazone or 3'-deamino3'(4-morpholinyl)- carminomycinN-substituted hydrazone and other compounds using the other derivatives,which are also within the scope of this invention.

The anthracycline derivatives of the present invention were evaluatedfor release rates of the drug adriamycin at pH values of 4.5, 5.0 and7.4 and exhibited a wide range of release rates. In addition, theimmunoconjugates comprising the derivatives conjugated to a monoclonalantibody were evaluated for release of adriamycin at a pH of 4.5. Theimmunoconjugates were tested for cytotoxicity using Daudi cells in theInhibition of Colony Formation assay and a correlation between thestability of the hydrazone derivatives was revealed. Theimmunoconjugates also demonstrated a wide range of release rates andexhibited antibody-directed cell killing (cytotoxicity) for tumor cellsin the colony formation assay.

The N-substituted hydrazine compounds of the invention provide usefulbifunctional compounds for linking molecules such as targeting andcytotoxic reagents. When used to link cytotoxic molecules containing acarbonyl group, the bifunctional compounds provide an acid-sensitivelinkage that is cleaved within a range of pH to release the cytotoxicreagent. The anthracycline immunoconjugates of this invention appear tobe an improvement over immunoconjugates reported previously, in whichanthracyclines were directly linked to antibodies through the aminosugar portion of the anthracycline, because these amino sugar-linkedconjugates often contain lower anthracycline to antibody molar ratios,are less potent than free ADM, and exhibit reduced antibody bindingproperties (Arnon et al., Immunological Rev. 62, supra; Hurwitz et al.,Cancer Res. 35, supra, and Yamamoto et al., supra). Furthermore,stability studies performed on the immunoconjugates of this inventionindicated that the anthracycline was released from the immunoconjugatesunder acidic conditions similar to those found in a cellularenvironment. Thus, the immunoconjugates of the present invention mayrelease relatively unmodified drug for delivery to the target cells. Theconjugates described herein provide release of drug at a wide range ofpH values which may be advantageous for drug delivery.

The bifunctional compounds, immunoconjugates of the invention and themethods for their production are exemplified by preferred embodiments inwhich derivatives of the anthracycline, adriamycin, were conjugated tothe antitransferrin receptor monoclonal antibody, 5E9.

The present invention also encompasses pharmaceutical compositions,combinations and methods for treating diseases such as cancers and othertumors, noncytocidal viral or other pathogenic infections, andautoimmune diseases. More particularly, the invention includes methodsfor treating disease in mammals wherein a pharmaceutically effectiveamount of at least one anthracycline-containing conjugate isadministered in a pharmaceutically acceptable manner to the host mammal.

Alternative embodiments of the methods of this invention include theadministration, either simultaneously or sequentially, of a number ofdifferent conjugates, i.e., bearing different cytotoxic reagents ordifferent antibodies or ligands, for use in methods of combinationchemotherapy. For example, an embodiment of this invention may involvethe use of a number of anthracycline-immunoconjugates wherein thespecificity of the antibody component of the conjugate varies, i.e., anumber of immunoconjugates are used, each one having an antibody thatbinds specifically to a different antigen or to different sites orepitopes on the same antigen present on the cell population of interest.The anthracycline component of these immunoconjugates may be the same ormay vary. For example, this embodiment may be especially useful in thetreatment of certain tumors where the amounts of the various antigens onthe surface of a tumor is unknown or the tumor cell population isheterogenous in antigen expression and one wants to be certain that asufficient amount of drug is targeted to all of the tumor cells at thetumor site. The use of a number of conjugates bearing differentantigenic or epitope specificities for the tumor increases thelikelihood of obtaining sufficient drug at the tumor site. Additionally,this embodiment is important for achieving a high degree of specificityfor the tumor because the likelihood that normal tissue will possess allof the same tumor-associated antigens is small (cf. Hellstrom et al.,"Monoclonal Antibodies to Two Determinants of Melanoma-Antigen p97 ActSynergistically In Complement-Dependent Cytotoxicity", J. Immunol., 127(No. 1) pp. 157-160 (1981)).

Alternatively, a number of different immunoconjugates can be used,wherein only the anthracycline component of the conjugate varies. Forexample, a particular antibody can be linked to adriamycin to form oneimmunoconjugate and can be linked to daunomycin to form a secondimmunoconjugate. Both conjugates can then be administered to a host tobe treated and will localize, due to the antibody specificity, at thesite of the selected cell population sought to be eliminated. Both drugswill then be released at that site. This embodiment may be importantwhere there is some uncertainty as to the drug resistance of aparticular cell population such as a tumor because this method allowsthe release of a number of different drugs at the site of or within thetarget cells. An additional embodiment includes the conjugation of morethan one type of anthracycline to a particular antibody to form animmunoconjugate bearing a variety of different anthracycline moleculesalong its surface, all linked to the antibody via a 13-keto hydrazonebond. Administration of the immunoconjugate of this embodiment resultsin the release of a number of different drugs at the site of or withinthe target cells. Furthermore, a combination of anthracycline-ligandconjugates can be used wherein the drug can be targeted to a cellpopulation carrying a specific antigen as well as a receptor for aspecific ligand on its surface. Again, one type of anthracycline or anumber of different drugs can be used in this combination therapy.

The conjugates of the invention can be administered in the form ofpharmaceutical compositions using conventional modes of administrationincluding, but not limited to, intravenous, intraperitoneal, oral,intralymphatic, or administration directly into the site of a selectedcell population such as a tumor. Intravenous administration ispreferred. In the case of the immunoconjugates, for in vivo treatment,it may be useful to use conjugates comprising antibody fragments such asFab or F(ab')₂ or chimeric antibodies.

The pharmaceutical compositions of the invention comprising theconjugates may be in a variety of dosage forms which include, but arenot limited to, solid, semi-solid and liquid dosage forms such astablets, pills, powders, liquid solutions or suspensions, suppositories,polymeric microcapsules or microvesicles, liposomes, and injectable orinfusible solutions. The preferred form depends upon the mode ofadministration and the therapeutic application.

The pharmaceutical compositions may also include conventionalpharmaceutically acceptable carriers known in the art such as serumproteins such as human serum albumin, buffer substances such asphosphates, water or salts or electrolytes.

The most effective mode of administration and dosage regimen for theconjugate compositions of this invention depends upon the severity andcourse of the disease, the patient's health and response to treatmentand the judgement of the treating physician. Accordingly, the dosages ofthe conjugates and any accompanying compounds should be titrated to theindividual patient. Nevertheless, an effective dose of the anthracyclineimmunoconjugate of this invention may be in the range of from about 1 toabout 100 mg/m² anthracycline or from about 500-5000 mg/m² antibody. Aneffective dose of the anthracycline-ligand conjugates may be in therange of from about 1 to about 100 mg/m² anthracycline or from about 1to about 100 mg/m² ligand.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the scope of this invention in anymanner.

EXAMPLES Preparation of Bifunctional Compounds and Derivatives ofAdriamycin (ADM)

Melting points (MP) were determined on a Fisher-Johns (Medford, Mass.)melting point apparatus and are uncorrected. Nuclear Magnetic Resonance(NMR) spectra were obtained on a Brucker AM 300 instrument. Infrared(IR) spectra were run as KBr pellets or CHCl₃ solutions on a P-E FTIR(Fourier Transformation Infrared) instrument (Norwalk, Conn.), model1800. MS (mass spectrum) and HRMS (high resolution mass spectrum) wereobtained with Kratos MS25RFA and MS50TC instruments (Manchester,England), respectively. Flash chromatography was performed using Woelm(Atlanta, Ga.) silica gel (silica 32-63). Thin Layer Chromatography(TLC) was carried out on Analtec (Newark, Del.) silica gel GHLF platesor RPS-F reversed phase plates, both 250 microns. For routine HighPressure Liquid Chromatography, (HPLC), a P-E pump, series 4 LC, a HP1046A fluorescence detector and a Phenomenex (Torrance, Calif.) IBSil-5C18 (150×4.6 mm) column were used. The mobile phase was a 70:30methanol-phosphate buffer (50 mmol ammonium phosphate, pH 4.4) at a flowrate of 1.5 ml/min. For release-rate measurements the HPLC systemcomprised of two Waters pumps model 510, an auto-sampler model 712 and agradient controller model 680. Chromatography was performed on a WatersC-18 column and the mobile phase was a 68:32 mixture of triethylammoniumformate buffer (0.05M, Ph 2.8) and acetonitrile, respectively. Thefluorescence of the eluted adriamycin was detected by using an ABIFluorescence Detector model 980 (excitation, 254 nm; emission, 550 nm)obtained from Applied Biosystems, Ramsey, N.J. Acetate buffer was usedfor pH 4.5 and 5.0; phosphate saline buffer was used for pH 7.4.Adriamycin-HCl was obtained from Sanraku Inc. (Japan). All otherchemicals were obtained from commercial sources. Elemental analyses wereperformed in the analytical department of Bristol-Myers Squibb Company,Wallingford, Conn. and at Oneida Research Services.

EXAMPLE 1 Preparation of Bifunctional Compound 10 and SemicarbazoneDerivative of ADM

The following example describes a method for producing a bifunctionalcompound and a semicarbazone derivative of ADM having a semicarbazonebond at the C-13 position of ADM. In this example,N-[2-[(2-pyridinyl)dithio)ethyl]hydrazinecarboxamide, compound 10, isprepared by the reaction sequence shown in FIG. 1. Reacting cysteaminehydrochloride with methoxycarbonylsulfenyl chloride followed by2-mercaptopyridine gave 2-(2-pyridinyl)dithioethanamine hydrochloride(compound 9 in FIG. 1). This in turn was reacted with phosgene andt-butyl carbazate followed by trifluoroacetic acid (TFA) to give thedesired product (compound 10).

Preparation of 2-(2-pyridinyl)dithio1ethanamine hydrochloride

A solution of methoxycarbonylsufenyl chloride (Zumach et al., AgnewChem. International Edit. 9:54-63 (1970), (6.33 g, 50 mmol) in HPLCgrade methanol (100ml) was stirred under N₂ and chilled in ice. To thisa solution of 2-aminoethanethiol hydrochloride (5.7 g, 50 mmol) inmethanol (50 ml) was added dropwise. After the addition was complete,the solution was stirred at room temperature (RT) for 2 h. The solventwas then evaporated and the residual oil crystallized from acetone (100ml) to yield a solid (6.9 g). This solid was dissolved in methanol (100ml). The solution was chilled in ice, stirred under N₂ and treateddropwise with a solution of 2-mercaptopyridine (3.82 g, 34 mmol) inmethanol (50 ml). The solution was stirred for 1 h at RT, concentratedto small volume and diluted slowly with acetone until crystallizationoccurred. After 1 h in the refrigerator the solid was collected byfiltration, and dried to yield the compound2-[(2-pyridinyl)dithio]ethanamine hydrochloride (compound 9). Thiscompound has been described by Field et al., J. Org. Chem. 29:1632-1635(1964) and Connor and Schroit, Biochem. 27:848-851 (1988)). The compoundwas characterized as follows: mp 123°-5° (5.8 g, 52%). IR (KBr) 2952,2913, 1610, 1575, 1559, 1451, 1115, 767 cm⁻¹. NMR (D₂ O). δ 8.46, 7.83,7.34 (d, m, m 4H, Py), 3.37 (t, 2H CH₂ CH₂ NH₂), 3.12(t, 2H, SCH₂ CH₂)MS(m/e): 187 (corresponds to [M+H]⁺) 170, 152, 142, 112, 104, 76.

Analysis: calculated for C₇ H₁₁ CLN₂ S₂.1/4 H₂ O: C, 37.00, H, 5.06, N,12.33. Found: C, 36.82: H, 4.99, N, 12.37.

Preparation ofN-[2-[(2-pyridinyl)dithio]ethyl]-2-(tert.butoxycarbonyl)hydrozinecarboxamide

2-[(2-pyridinyl)dithio]ethanamine hydrochloride (2.22 g, 10 mmol) wassuspended in dry methylene chloride (100 ml) and treated withtriethylamine (TEA) (5.8 ml). This solution was added dropwise to an icecold stirred solution of phosgene (10 ml of a 1.93 M toluene solution)in methylene chloride (200 ml). The reaction was monitored by TLC andwhen no more starting material was present, N₂ was passed throughout themixture for a while. Then t-butyl carbazate (1.32 g, 10 mmol) was addedand the mixture left to stir overnight. The solution was washed withwater and the solvent evaporated. The residue was chromatographed oversilica using methylene chloride:methanol (100:2) solvent system. Theappropriate fractions were combined to yield 1.74 g ofN-[2-[(2-pyridinyl)dithio]ethyl]-2-(tert-butoxycarbonyl)hydrazinecarboxamide(compound 9a) as a foam, characterized as follows:IR (KBr) 3281, 2979, 2932, 1723, 1672, 1577, 1560, 1545, 1448, 1419,1253, 1161, 762 cm⁻¹. NMR(CDCl₃)δ 8.50, 7.56, 7.51, 7.12(4H, Py),3.51(2H, CH₂), 2.89(2H, CH₂ S), 6.84, 6.37, 6.17(3H, NH), 1.44(9H),(CH₃)₃ C). MS(m/e) 345 (corresponds to [M+H]⁺), 317, 289, 245, 213, 178,134, 112.

Preparation of N-[2-[(2-pyridinyl)dithio1-ethyl]hydrazinecarboxamide.

N-[2-[(2-pyridinyl)dithio]ethyl]-2-(tertbutoxycarbonyl)hydrazinecarboxamide(570 mg, 1.66 mmol) was dissolved in ice cold TFA (10 ml). The solutionwas stirred in ice for 10 min and additional 10 min without cooling. Theexcess of TFA was evaporated under reduced pressure as much as possibleand the residue chromatographed over silica using amethylene-chloride:methanol:concentrated ammonium hydroxide (100:5:0.5)solvent system. The appropriate fractions were combined according to TLCand the solvent evaporated to leave a crystalline residue (0.42 g,quantitative). An analytical sample was prepared by crystallizing fromIPA, mp 105°-7°. N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide(compound 10) was characterized as follows: IR(KBr) 3336, 3220, 3064,2949, 2934, 1670, 1623, 1575, 1562, 11-33, 1452, 1369, 1172, 1046, 770cm⁻¹. NMR(CD₃ OD)δ 8.41, 7.78, 7.21(4H, Py), 3.43(2H, NCH₂), 2.91(2HSCH₂). MS(m/e) 245(corresponds to [M+H]⁺ 0, 221, 213, 162, 134, 112.

Analysis: Calculated for C₈ H₁₂ N₄ OS₂ : C, 39.32, H, 4.95; N, 22.93; S,26.24. Found, C 39.19; H, 4.86; N, 22.48; S, 25.02.

Preparation of the semicarbazone derivative of adriamycin hydrochlorideand N-[2-(2-pyridinyl)dithio]ethyl] hydrazinecarboxamide.

Compound 10 (0.37 g, 1.5 mmol) in methanol (25 ml) was added to astirred suspension of adriamycin hydrochloride (0.66 g, 1.14 mmol) inmethanol (50 ml). TFA (5 drops) was added and the mixture left to stirovernight. The clear solution was concentrated and chromatographed overa C-18 column using methanol:water (60:40) containing 0.3% ammoniumacetate, as solvent system. The appropriate fractions were combined andthe methanol evaporated as much as possible. The aqueous phase wasfreeze-dried and the residue dissolved in methanol and added toacetonitrile. The red solid was collected by centrifugation, and dried.(0.65 g, 68%). The semicarbazone derivative of ADM was characterized asfollows: IR(KBr) 3399, 2976, 2936, 1671, 1618, 1578, 1538, 1417, 1286,1210, 1117, 1015, 989, 764 cm⁻¹. NMR(CD₃ OD) δ 8.25, 7.76, 7.62, 7.48,7.07(py, ph, H), 4.95(anomeric H), 4.63(CH₂ OH), 4.24(CH₃ CH),3.97(OCH₃), 3.5-2.9 (cluster absorption of SSCH.sub. 2, --CH₂ --, CH₂--NH), 1.29 (HC--CH₃). MS(m/e) 770 (corresponds to [M+H]⁺), 641, 437,346. HRMS: calculated C₃₅ H₄₀ N₅ O₁₁ S₂ : 770.2166; Found: 770.2157.

EXAMPLE 2 Preparation of Bifunctional Compound 11a and the CarbazoneDerivative of ADM

The following example describes a method for producing a carbazidebifunctional compound and the carbazone derivative of ADM having acarbazone bond at the C-13 position of ADM. In this example, thereaction shown in FIG. 2 and described in Example 1 between2-[(2-(pyridinyl)dithio]ethanamine hydrochloride and t-butyl carbazatewas started with t-butyl carbazate and triphosgene, as shown in FIG. 3,to yield a bifunctional compound, (compound 11a), a carbazide. In thiscase, excess t-butyl carbazate was reacted with phosgene to give thecarbonic dihydrazide.

Preparation of 2-[[2-[(2-pyridinyl)dithio]ethyl] amino]carbonyl]-2,2'-bis(tert.-butoxycarbonyl) carbonic dihydrazide.

t-butyl carbazate, (0.396 g, 3 mmol) was dissolved in dry chloroform (10ml). The solution was stirred under N₂ at RT and TEA was added (0.6 g, 6mmol). This was followed by the addition of triphosgene (0.296 g, 1mmol) all at once. A vigorous reaction ensued and when it subsided,2-(2-pyridinyldithio)ethanamine hydrochloride (0.667g, 3 mmol) inchloroform containing TEA (0.3 g, 3 mmol) was added. The mixture wasstirred at RT for 11/2 h then washed with water (3×20 ml) dried and thesolvent evaporated under reduced pressure to leave a foam (0.91 g). Thismaterial was chromatographed over silica using methylenechloride:methanol (100:2) solvent system. The fractions were monitoredby TLC and combined accordingly to yield the compound2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]-2,2'-bis(tert.-butoxycarbonyl)carbonic dihydrazide (compound 11) as a foam (0.54 g, 52%). Compound 11was characterized as follows: IR(KBr)3302, 2980, 2933, 1726, 1683, 1498,1252, 1160, 1047, 1018, 763 cm⁻¹. NMR(CDCl₃) δ 8.50, 7.57, 7.49, 7.10,(d,q,d,t, 4H, Py), 3.52 (t, 2H, SSCH₂) 2.90 (t, 2H CONCH₂), 1.46[C(CH₃)₃ ], 8.30, 6.50, 6.29 (b,s,s, NH}. MS (m/e) 503 (corresponds to[M+H]⁺), 447, 431, 419, 403, 347, 303, 213, 179, 112.

Preparation of 2-[[[2-[(2-pyridinyl)dithio]ethyl] amino]carbonylcarbonic dihydrazide

Compound 11 (0.34 g, 0.68 mmol) was stirred for 10 min with ice cold TFA(5 ml) and an additional 10 min without cooling. The TFA was evaporatedas much as possible and the residue was chromatographed over silicausing methylene chloride:methanol:concentrated NH₄ OH (100:5:0.5)solvent system. The appropriate fractions were combined and afterevaporation compound 11a was obtained as a hygroscopic foam (0.2 g,quantitative yield). Compound 11a was characterized as follows: IR(film) 3330, 2964, 2929, 1698, 1660, 1576, 1486, 1231, 1045, 759 cm⁻¹.NMR (CDCl₃) δ 8.50, 7.56, 7.10 (d,m,m,4H, Py), 3.52 (q, 2H, CH₂ N), 2.91(t, 2H, CH₂ SS), 8.87, 8.85, 4.19, 3.78 (D₂ O exchangeable protons, NH).MS (m/e) 303 (corresponds to [M+H]⁺), 213, 112.

Preparation of the carbazone derivative of adriamycin hydrochloride and2-[[[2-[(2-pyridinyl)dithio]ethyl] amino]carbonyl]carbonic dihydrazide

Adriamycin hydrochloride (356 mg, 0.6 mmol) and compound 11a (0.2 g,0.68 mmol) were left to stir overnight in methanol (50 ml) containing2-3 drops of TFA. A clear solution was obtained and HPLC (methanol: 0.01M ammonium phosphate solution, pH 4.5, 70:30 solvent system) indicatedthat over 90% of the adriamycin had been converted to the semicarbazone.The solvent was therefore evaporated and the residue chromatographedover a C-18 column using a solvent system of methanol:water (60:40) andcontaining 0.3% ammonium acetate. The fractions were monitored byreversed phase TLC (same solvent system but 3% ammonium acetate) and/orHPLC and fractions free of adriamycin were combined. Most of themethanol was evaporated under reduced pressure. The aqueous solution wasfreeze-dried and the red residue was dissolved in a small volume ofmethanol. The solution was filtered and added to stirred acetonitrile (1L). The clear solution was concentrated to about a third of its volumeand the solid obtained was collected by centrifugation and dried to givethe carbazone of ADM (Compound 4) (160 mg). A second crop (85 mg) wasobtained by concentrating the solution to 100 ml, diluting with etherand collecting the solid by centrifugation (total yield 49%). Thiscarbazone derivative was characterized as follows: IR(KBr): 3346, 2975,2936, 1711, 1668, 1618, 1578, 1286, 1210, 1083, 1015, 765 cm⁻¹. NMR(CD₃OD) δ 8.43, 7.89, 7.77, 7.52, 7.21, (Py, phenyl H), 5.15 (anomeric H)4.57 (CH₂ OH), 4.25 (CH₃ CH), 3.99 (OCH₃), 3.53 (SSCH₂), 3.17 (--CH₂ --,ring), 3.05 (CH₂ NN═), 2.38 (--CH₂ --, ring) 1.29 (CHCH₃). MS(m/e) 828(corresponds to [M+H]⁺), 699, 572, 537, 377, 346, 289, 213.

EXAMPLE 3 Preparation of Bifunctional Compound 12 and theThiosemicarbazone derivative of ADM

This example describes the preparation of a bifunctional compound,compound 12, and the thiosemicarbazone derivative of ADM having athiosemicarbazone bond at the C-13 position of ADM. In this example, thethio analog of the semicarbazide described above in Example 1 (compound1 0) was prepared as shown diagrammatically in FIG. 4. When using2-[(2-pyridinyl)dithio]ethanamine, an elimination of 2-mercaptopyridinewas observed which could be ascribed to the increased nucleophilicity ofthe thiosemicarbazide moiety in the penultimate product stage. Thisproblem was circumvented by employing the trans-2-butene group as shownin FIG. 4.

Preparation of 1-bromo-4-(N-phtalimido)-2-butene.

To a solution of 1,4-dibromo-2-butene (8.4 g, 40 mmol) in DMF (200 ml)was added potassium phthalimide (4.62 g 24 mmol) portionwise during 1 h.After stirring overnight, the solvent was evaporated and the residuepartitioned between water and methylene chloride. The organic layer waswashed several times with water, dried and the solvent evaporated. Theresidue was crystallized from 2-propanol yielding the desired product1-bromo-4-(N-phtalimido-2-butene) (3.95 g, 59%); characterized asfollows: mp 101°-2°. IR(KBr) 1775, 1711, 1466, 1436, 1393, 723 cm⁻¹. NMR(CDCl₃) δ 7.81, 7.73 (m,m 4H, phenyl), 5.88, 5.81 (m,m 2H, 2 ═CH--)4.30(d,2H CH₂ --N), 3.90 (d,2H CH₂ Br). MS (m/e) 280 (corresponds to[M+H]⁺), 200.

Analysis: Calculated for C₁₂ H₁₀ BrNO₂ : C,51.45; H, 3.60; N, 5.00.Found: C, 52.35; H, 3.47, N, 4.80.

Preparation of 1-(acetylthio)-4-(N-phthalimido)-2-butene

A mixture of 1-bromo-4-(N-phthalimido)-2-butene (3.95 g; 14 mmol) andpotassium thioacetate (1.77 g, 15.5 mmol) in absolute ethanol (50 ml)was heated to reflex for 1/2 h. The solvent was evaporated and theresidue was extracted with methylene chloride. The solvent wasevaporated to yield a crystalline residue (3.85 g, 99%) which was usedas such for the next step. An analytical sample was prepared bycrystallization from 2-propanol, mp 69°-71°. This compound wascharacterized as follows: IR(KBr) 1769, 1713, 1688, 1427, 1391, 1114,958 cm⁻¹. NMR (CDCl₃) δ 7.80, 7.73 (m,m 4H Ph), 5.70 (m, 2H, 2 ═CH--),4.24 (d, 2H, CH₂ N), 3.48 (t, 2H CH₂ S), 2.29 (s, 3H, C--CH₃). MS (m/e)276 (corresponds to [M+H]⁺), 234, 200.

Analysis: Calculated for C₁₄ H₁₃ NO₃ S: C, 61.07; H, 4.76; N, 5.09.Found: C, 61.29; H, 4.82, N,5.21.

Preparation of 1-amino-4-(2-pyridinyl)dithio]-2-butene hydrochloride

A solution of 1-acetylthio-4-(N-phtalimido)-2-butene (6.5 g, 23.6 mmol)in absolute ethanol (150 ml) and hydrazine (1.74 g, 54 mmol) was heatedto reflux. The reaction was followed by TLC and when no startingmaterial was present the solution was chilled in ice and treated with 6NHCl (10 ml). A voluminous precipitate formed and was identified asphtalhydrazide (NMR, MS) and it was filtered off. The filtrate wasconcentrated to 10 ml and diluted with water. The solid was filtered offand the filtrate was washed with ether (2X) and methylene chloride (1X),filtered through Celite and freeze-dried. The solid was dissolved in asmall amount of methanol and the solution was filtered through Celite,the solvent evaporated and the residue evacuated overnight. A waxy,hygroscopic material was obtained, having the characteristics of: NMR(DMSO-D₂ O) δ 5.83, 5.60 (m,m 2H, 2 ═CH--) 3.40 (d, 2H CH₂ NH₂), 3.16 (d2H, CH₂ SH) MS (m/e) 104 (corresponds to [M+H]⁺), 87, 70. This waxymaterial was dissolved in HPLC grade methanol (75 ml). The solution wasstirred and treated with methoxycarbonylsulfenyl chloride (3 g, 23.7mmol). After 1/2 h starting material was not detected by TLC. Thesolvent was evaporated and the residue was redissolved in methanol (75ml). The solution was stirred and treated with 2-mercaptopyridine (2.7g, 24 mmol). After 2 h the solvent was evaporated and the residueevacuated at high vacuum. The residue was then dissolved in a mixture of0.01 N HCl and methanol (90:10, 130 ml). The cloudy solution was washedwith methylene chloride, filtered through Celite and freeze-dried toyield 1-amino-4-[(2-pyridinyl)dithio]-2-butene hydrochloride as a highlyhygroscopic fluffy material (4 g, 68%) having the characteristics ofIR(KBr) 3433, 2959, 2884, 1607, 1576, 1447, 1418, 1118, 767 cm⁻¹. NMR(D₂O) δ 8.51, 8.13, 8.02, 7.54 (m 4H, Py) 5.87, 572 (m,m 2H, 2 ═CH--) 3.54(d 2H, CH₂ NH₂) 3.43 (d 2H, CH₂ S). MS (m/e) 213 (corresponds to[M+H]⁺), 196, 112.

Preparation of N-[4-[(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide

1-amino-4-[(2-pyridinyl)dithio]-2-butene hydrochloride (1.5 g., 6 mmol)was suspended in stirred methylene chloride (30 ml). TEA Was added (1.46g, 14.6 mmol) followed by di-2-pyridyl thionocarbonate (Kim and Yi,Tetrahedron Lett. 26:1661-1664 (1985)), (1.4 q, 6 mmol). A clearsolution was obtained and TLC showed the absence of starting material.t-Butyl carbazate (0.8 g, 6 mmol) was added and the solution stirred for1 h. The solution was washed with water and the solvent evaporated. Theresidue was chromatographed over silica using methylenechloride:methanol (100:2) solvent system and rechromatographed usinghexane:ethyl acetate (75:25) solvent system, to yield a foam which isthe t-boc derivative of N-[4-[(2pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide (1.4 g, 58follows:IR(KBr) 3238, 2971, 2930, 1718, 1544, 1418, 1156, 762 cm⁻¹. NMR (CDCl₃/D₂ O) δ 8.43, 7.65, 7.08 (m, m, m 4H, py) 5.57 (m 2H, 2═CH), 4.12 (d2H, CH₂ N) 3.45 (d 2H CH₂ S), 1.46 (s 9H, 3 CH₃). MS (m/e) 387(corresponds to [M+H]⁺) 355, 287, 276, 112.

The protected carbothioamide (0.86 g, 2.2 mmol) was dissolved in icecold TFA. The solution was kept in ice for 10 min (under nitrogen) andfor an additional 10 min without cooling. The excess acid was evaporatedas much as possible at high vacuum and the residue was chromatographedover silica using a methylene chloride:methanol:concentrated NH₄ OH(100:5:0.5) solvent system. The appropriate fractions were combined toyield compound 12 in the form of a gum (0.39 g, 63%), having thecharacteristics of: IR(film) 3322, 3198, 2974, 1626, 1574, 1560, 1538,1448, 1418, 1224, 760 cm⁻¹. NMR (CDCl₃ /D₂ O) δ 8.41, 7.63, 7.11 (m m m4H, py) 5.64 (m 2H, 2═CH), 4.20 (d 2H, CH₂ N) 3.41 (d 2H, CH₂ S). MS(m/e) 287 (corresponds to [M+H]⁺), 225, 221, 144, 112.

Preparation of the thiosemicarbazone derivative of adriamycinhydrochloride and N-4-(2-pyridinyl)dithio]-2-butenyl]hydrazinecarbothioamide

To a stirred suspension of adriamycin hydrochloride (350 mg, 0.6 mmol)in HPLC grade methanol (50 ml) a solution of compound 12 (350 mg, 1.2mmol) in HPLC grade methanol (25 ml) was added. TFA (3-4 drops) wasadded and the mixture was left to stir overnight. A clear solution wasobtained and no free adriamycin was detected either by HPLC or TLC. Thesolution was concentrated to a small volume (5 ml) which was added toacetonitrile (600 ml). A precipitate formed and after cooling in therefrigerator was collected by centrifugation and dried at high vacuum(275 mg, 54%). The thiosemicarbazone derivative was characterized asfollows: IR(KB_(r)) 3418, 2934, 1616, 1578, 1534, 1414, 1284, 1208,1012, 986 cm⁻¹. NMR(CD₃ OD) δ 8.30, 7.80, 7.74, 7.52, (Py, phenyl 7H),5.60 (2═CH) 5.40 (anomeric H) 4.67 (CH₂ OH), 4.22 (CH₃ CH) 4.09 (CH₂ N)4.02 (OCH₃) 3.5-1.88 cluster absorption including--CH₂ --SS,--CH₂ --CH),1.30 (CH₃ --CH). MS (m/e) 812 (corresponds to [M+H]⁺), 701, 683, 669,572, 554, 540, 536, 522, 504. HRMS Calculated for C₃₇ H₄₂ N₅ O₁₀ S₃ :8.12.2094; Found 812.2087.

EXAMPLE 4 Preparation of Bifunctional Compound 13 and theCarboxylatehydrazone Derivative of ADM

This example describes the preparation of novel bifunctional compound 13and the carboxylatehydrazone derivative of ADM. The carboxylatehydrazinebifunctional compound is prepared starting with mercaptoethanol whichwas derivatized to pyridinyldithioethanol as shown in FIG. 5. Thiscompound was then converted to an activated carbonyl derivative that wascondensed with hydrazine.

Preparation of 2[(2-(pyridinyl)dithioethyl hydrazine carboxylate

To a cooled (0° C.) solution of chlorocarbonylsulfenyl chloride (1.24 g,9.45 mmol) in CH₂ Cl₂ (10 ml) was added dropwise 2-mercaptoethanol, (737mg, 9.45 mmol). The mixture was stirred for 30 min at 0°-15° C., cooledto 0° C. and treated with a solution of 2-mercaptopyridine (1.05 g, 9.45mmol) in CH₂ Cl₂ (15 ml). The mixture was stirred at 0° C. for 1 h andthen at room temperature for 16 h. After addition of an ammoniumcarbonate solution (1.0 g in 20 ml H₂ O), the layers were separated andthe organic layer was washed with water, dried, and concentrated invacuo to give crude 2-(2-pyridinyldithio)ethanol (1.75 g) as a colorlessoil. Carbonyldiimidazole (648 mg, 4 mmol) was added to a solution of2-(2-pyridinyldithio)ethanol (714 mg, 3.8 mmol) in CH₂ Cl₂ (10 ml). Themixture was stirred for 20 h and then cooled to -20° C. and treated withhydrazine (122 mg, 3.8 mmol). The mixture was left standing at -5° C.for 16 h, and then concentrated in vacuo. The residue waschromatographed on silica gel using a methylenechloride:methanol(100:1-3) solvent system to give compound 13,2-[(2-pyridinyldithio) ethyl hydrazinecarboxylate (340 mg, 37%), as acolorless oil, having the characteristics as follows: NMR (CDCl₃) δ 8.46(1H), 7.62 (2H), 7.08 (1H), 5.92 (1H), 4.35 (t, 2H), 3.70 (s, ZH), 3.01(t, 2H), 1.56 (s,2H). MS (m/e) 246 (corresponds to [M+H]⁺), 142, 103.

Preparation of the hydrazone derivative of adriamycin hydrochloride and2[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate

To a suspension of adriamycin hydrochloride (290 mg, 0.5 mmol) inanhydrous methanol (4 ml) were added a solution of compound 13 (170 mg,6.9 mmol) in methanol (4 ml) and CF₃ CO₂ H (6 mg) in methanol (1 ml).After stirring for 24 h the mixture was concentrated to about 4 ml andacetonitrile (50 ml) was added to this solution. The product wasisolated by centrifugation. The solid was dissolved in water-methanoland then lyophilized to give the hydrazone derivative of adriamycin,(354 mg, 88%) as a dark red solid, having the characteristics asfollows: NMR (CD₃ OD) δ 8.34 (1H), 7.93 (d, 1H), 7.81 (m, 3H), 7.54 (d,1H), 7.17 (m, 1H), 5.49 (m, 1H), 5.19 (s, 1H), 4.59 (m,2H), 4.37 (m,2H), 4.25 (m, 1H), 4.01 (s, 3H), 3.63 (m, 1H), 3.54 (m, 1H), 3.10 (t,3H), 2.37 (m,2H), 2.03 (m, 1H), 1.89 (m,1H), 1.29 (d,3H}. MS (m/e): 771(corresponds to [M+H]⁺), 642.

EXAMPLE 5

Preparation of Bifunctional Compound 15 and the Arylhydrazone Derivativeof ADM

This example describes the method for preparing a novel bifunctionalcompound, compound 15, and the arylhydrazone derivative of ADM having anarylhydrazone bond at the C-13 position of ADM. Compound 15 is preparedusing 4-N-Boc-hydrazinobenzoic acid, and incorporating the2-(2-pyridinyldithio)ethanamine group as shown in FIG. 6.

4-(N-boc-hydrazino)benzoic acid

p-Hydrazinobenzoic acid (760 mg, 5 mmol) was dissolved in dioxane (10ml), water (5 ml), and 1 N NaOH solution (5 ml). Di-t-butylpyrocarbonate(1.31 g, 6 mmol) was added at 0° C. and the reaction mixture was stirredat 0° C. for 1 h and at RT for 30 min. After this period the volume ofthe solution was reduced to a half and the solution was acidified with0.5% HCl solution and extracted with EtOAc. The combined EtOAc solutionwas washed with brine and dried over Na₂ SO₄. Removal of the solventgave a slightly brown solid which was recrystallized from EtOAc andhexane (950 mg, 75%), and was characterized as follows: NMR (CD₃ OD) δ7.84 (d, 2H, J=8.5 Hz), 6.75 (d, 2H, J=8.5 Hz), 1.4s (s, 9H); IR (KBr)3316, 1688, 1607, 1298 cm⁻¹.

Preparation ofN-[2-[(2-pyridinyl)dithio]ethyl-4-(N-bochydrazino)benzamide acid

4-(N-boc-hydrazino)benzoic acid (252 mg, 1 mmol) N-hydroxysuccinimide(115 mg, 1 mmol), and dicyclohexylcarbodiimide (DCC) (247 mg, 1.2 mmol)in N,N-dimethylformamide (DMF) (5 ml) were stirred overnight at roomtemperature. Dicyclohexylurea (DCU) was filtered off and the filtratewas evaporated. The residue was crystallized by addition of Et₂ O togive the N-hydroxysuccinimide ester of 4-(N-boc-hydrazino)benzoic acid(300 mg). This material (250 mg, 0.72 mmol) and2-(2-pyridinyl)dithio)ethylamine hydrochloride (167 mg, 0.75 mmol) weredissolved in DMF (4 ml). After addition of TEA (0.125 ml, 0.9 mmol), themixture was stirred overnight at room temperature. DMF was removed andthe residue was chromatographed on SiO₂ (2% MeOH-CH₂ Cl₂) to give a foam(2.17 mg, 52%), having the characteristics as follows: NMR (CDCl₃) δ8.37 (d, 1H, J=5.1 HZ), 8.01 (bt, 1H), 7.78 (d, 2 H, J=8.7 Hz), 7.57 (m,1H), 7.46 (d, 1H, J=8.1 Hz), 7.09 (m, 1H), 6.84 (d, 2H, J=8.7 Hz), 7.40(bs, 1H), 5.92 (bs, 1H), 3.70 (m, 2H), 2.98 (t, 2H, J=5.8 Hz), 1.45 (s,9H); IR (KBr) 3303, 1714, 1610, 1505 cm⁻¹ ; ms m/e 421 (M+H), 365, 321,112, HRMS calculated for C₁₉ H₂₅ N₄ O₃ S₂ 421.1368, found 421.1358.

Preparation of N-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide

The previous compound (200 mg, 0.48 mmol) was treated with TFA (1.5 ml)at 0° C. for 1 h. After this period TFA was evaporated and the residuewas triturated with Et₂ O to give approximately 200 mg ofN-[2-[(2-pyridinyl)dithio]ethyl]-4-hydrazinobenzamide (compound 15) inthe form of an oil having the characteristics of: NMR (CD₃ OD) δ 8.38(d,1H, J=4.5 Hz), 7.81 (m, 4H), 7.22 (t, 1H, J=5.8 Hz), 6.97 (d, 2H,J=8.8 Hz), 3.67 (t, 2H, J-6.6 Hz), 3.06 (t, 2H, J=6.6 Hz); IR (film)3278, 1674, 1613 cm⁻¹ ; MS m/e 321 (M+H).

Preparation of arylhydrazone derivative of adriamycin andN-[2-[(2-pyridinyldithio)ethyl]-4-hydrazinobenzamide

Compound 15 and adriamycin hydrochloride (250 mg, 0.43 mmol) weredissolved in MeOH (15 ml) and stirred in the dark for 2 days. Thesolvent was removed and the residue was chromatographed on C-18 reversedphase SiO₂. Elution with MeOH:H₂ O=2:1 containing 0.3% NH₄ OAc gavehydrazone compound 7 as orange powder (30 mg, 8%), having thecharacteristics as follows: NMR (CD₃ OD) δ 8.33 (d, 1H, J=4.8 Hz), 7.85(d, 1H, J=7.9 Hz), 7.74 (t, 1H, J=8.0 Hz), 7.66 (m, 4H), 7.41 (d, 1H,J=8.5 Hz), 7.14 (m, 1H), 7.02 (d, 2H, J=8.8 Hz), 5.46 (bs, 1H), 5.16 (m,1H), 4.60 (s, 2H), 4.23 (m, 1H), 3.91 (s, 3H), 3.62 (m, 4H), 3.02 (m,4H), 2.61 (m,1H), 2.38 (m, 1H), 1.97 (m,2H), 1.32 (d, 3H, J=6.5 Hz); IR(KBr) 3206, 1708, 1607, 1578 cm⁻¹ ; MS m/e 846 (M+H), 737, 717, HRMScalculated for C₄₁ H₄₄ N₅ O₁₁ S₂, 846.2479, observed 846.2380.

Elution with MeOH:H₂ O=3:1 containing 0.3% NH₄ OAc gave the anhydroderivative as a blue solid (120 mg, 34%). NMR (CD₃ OD) δ 8.40 (d, 1H,J=4.1 Hz), 7.77 (m, 5H), 7.42 (m, 1H), 7.22 (m, 1H), 7.16 (m, 2H), 5.35(bs, 1H), 5.26 (m,1H), 4.65 (s, 2H), 4.00 (m, 1H), 3.93 (s, 3H), 3.67(t, 2H, J=6.5 Hz), 3.44 (m, 1H), 3.08 (t, 2H, J=6.5 Hz), 2.49 (m, 1H),1.88 (m, 1H), 1.63 (m, 1H), 1.19 (d, 3H, J=6.5 Hz); MS m/e 828 (M+H)699, 681, 495; HRMS calculated for C₄₁ H₄₁ N₅ O₅ S.sub. 2 828.2372,observed 828.2300.

EXAMPLE 6 Characterization of the ADM Derivatives

The release of ADM from the ADM derivatives of the invention prepared asdescribed in Examples 1-5 above, at various pH's, ranging from 4.5 to7.4, was studied using HPLC analysis. Stock solutions (1 mg/ml) of theADM-derivatives were prepared in methanol and aliquots were diluted intoaqueous buffer solution at pH 4.5, 5.0 and 7.4 to achieve finalconcentrations of approximately 1.6 nmol/ml. Incubations in each bufferwere carried out at 37° C. for up to 24 h and the aliquots were analyzedby application to an HPLC column to determine the amount of unconjugatedADM. The released material was identified as intact ADM by its retentiontime on the column and by a UV profile of the eluted material. Therelease rates were expressed as a percentage of the maximum amount ofADM and are shown in FIGS. 10-12.

As illustrated in the figures, the ADM derivatives of the inventiondemonstrated wide-ranging release rates. The amount of material releasedfrom the ADM-derivatives increased as the pH was lowered from 7 to 4.The ADM-derivatives have an acid-sensitive linkage group which resultsin release of ADM from the antibody protein. These results areconsistent with the existence of a semicarbazone, carbazone,thiosemicarbazone, carboxylatehydrazone or arylhydrazone bond joiningthe ADM to the bifunctional compound.

EXAMPLE 7 Preparation of Anthracycline Immunoconjugates

This example describes the preparation of anthracycline immunoconjugatesaccording to the present invention wherein the above-described ADMderivatives (Examples 1-5) are conjugated to a monoclonal antibody.

Preparation of Immunoconjugates Having a Disulfide Bond Within theBifunctional Compound

The monoclonal antibody used was 5E9 produced from hybridoma ATCC No.HB21, available from the American Type Culture Collection "ATCC" inRockville, Md. Monoclonal antibody 5E9 is an IgG₁ antibody reactive withthe transferrin receptor on all dividing human cells and cross-reactivewith various histological types of cancer cells. 5E9 was purified fromascitic fluid produced in BALB/c mice according to the procedure ofBruck et al., "One-Step Purification of Mouse Monoclonal Antibodies FromAscitic Fluid by DEAE-Affigel Blue Chromatography" J. Immunol. Methods5b:313-319 (1982)).

Before reacting the ADM-derivative with the monoclonal antibodyselected, the antibody was thiolated, i.e. to introduce reactivesulfhydryl groups onto the antibody molecule. Thiolation of the 5E9monoclonal antibody (MAb) was performed using SPDP essentially asdescribed by Greenfield et al., supra. Briefly, SPDP (Pierce ChemicalCo., Ill.) (50 mM), dissolved in ethanol, was added to the 5E9 MAb (5-10mg/ml) in phosphate buffered saline (PBS), pH 7.2, to give a finalconcentration of between 5-10 mM. The reaction mixture was incubated for30 min at 30° C. Unreacted SPDP was separated from SPDP-derivatizedantibody by gel filtration chromatography using a PD-10 column(Pharmacia). The reactive pyridinyldithio moieties were removed byreduction with excess DTT. The reduced antibodies were passed through aPD-10 column and the free thiol-containing antibodies were used forcondensation with the ADM derivatives.

Reactive thiol groups were also introduced onto the antibody proteinusing 2-IT. The antibody (5-10 mg/ml in 50 mM TEA, 50 mM NaCl, 1 mM EDTAat pH 8.0) was mixed with 2-IT (Pierce Chemical Co., IL) at a finalconcentration of 5-10 mM. The reaction was allowed to proceed for 90 minat 4° C. and thiolated antibodies were separated on a PD-10 columnequilibrated with 2 M NaCl/PBS.

The number of reactive thiol groups incorporated onto the antibody wasdetermined using DTNB (5,5'-dithiobis(2-nitrobenzoic acid) (E₄₁₂₌ 14150)according to the procedure described by Ellman, Arch. Biochem. Biophys.82:70-77 (1959)).

Each ADM-derivative was dissolved in DMF and added to the reducedSPDP-thiolated MAb 5E9 in PBS. The amount of ADM-derivative wasequivalent to the number of thiol groups on the antibody. Theconjugation reaction was allowed to incubate overnight at 4° C. Afterthis period the antibody solution was dialyzed against PBS to removeunconjugated adriamycin derivative. The antibody solution was thentreated with SM-2 BioBeads (Bio-Rad Laboratories, Richmond, CA)overnight. The amount of conjugated anthracycline bound to the MAb wasdetermined by absorbance at 495 nm (E₄₉₅ =8030). The amount of antibodyprotein was determined by absorbance at 280 nm (1 mg/ml=1.4 O.D. units).To correct for the overlap of ADM absorbance at 280 nm, the followingformula was used: ##EQU1##

Immunoconjugates were analyzed for the presence of unconjugated ADM orADM derivatives using HPLC analysis. HPLC was done using a Phenomenexcolumn packed with 5 micron IB-SIL C18 beads. Unconjugated drug,ADM-derivatives (i.e. ADM conjugated to each of the bifunctionalcompounds of the invention prepared as described in Examples 1-5 above)(0.1 μmoles), or immunoconjugates containing 0.5-5 μmoles drugequivalents were applied to the column and eluted with methanol and 10mM ammonium phosphate, pH 4.5 (70:30) at 1.5 ml/min. Theimmunoconjugates produced contained no significant amount (less than 1%)of unconjugated drug as determined by HPLC analysis.

EXAMPLE 8 Characterization of the Immunoconjugates

The immunoconjugates produced as described above in Example 7 werecomprised of ADM molecules conjugated at the 13-keto position to abifunctional compound that formed a link between the ADM and MAb 5E9.Furthermore, the addition of the MAb with free thiol groups to the ADMderivative which contained a reactive pyridinyldithio moiety, led to theformation of a disulfide bond in the bifunctional compound joining ADMto the MAb. Immunoconjugates formed according to this embodimentinclude, but are not limited to, 5E9-ADM-semicarbazone-[3.42];5E9-ADM-carbazone-[4.37thiosemicarbazone-[2.51];5E9-ADM-carboxylatehydrazone-[2.35]; and 5E9-ADM-arylhydrazone-[2.52],wherein the first part of the designation represents the monoclonalantibody used to form the conjugate, the second part represents theanthracycline linked to the antibody and the numeral in the designationrepresents the molar ratio of ADM/antibody in the particular conjugate.

The binding activity of the immunoconjugates of the invention wasdetermined in a fluorescence binding assay as described by Greenfield etal., in "In Vitro Evaluation of Immunoconjugates Prepared by LinkingMitomycin C to Monoclonal Antibodies via Polyglutamic Acid Carriers" inAntibody Immunoconjugates and Radiopharmaceuticals, Vol. 2, p. 201(1989). Briefly, the immunoconjugates were serially diluted into 100 μlassay media (RPMI 1640 supplemented with ;0% fetal calf serum andpenicillin/streptomycin, Gibco, Grand Island, N.Y.). CEM tumor cells(ATCC No. CCL 119) (1×10⁶ cells) grown in the same medium were harvestedand washed by centrifugation and then suspended (1×10⁶)in the mediumcontaining the diluted immunoconjugates. After one hour of incubation at4° C., cells were washed and suspended in 100 μl medium containing 1:40diluted goat anti-mouse IgG-FITC (Cappel, Durham, N.C.) for additionalone hour at 4° C. Cells were washed and analyzed using a Coulter Epics Vfluorescence cell analyzer. For each experiment, similarly diluted MAbwas used as a non-conjugated positive binding control. The percentage ofprotein yield (obtained separately), molar ratios (moles of ADM/MAb) andbinding expressed as the percentage of original binding are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                        5E9 Immuno-  Protein   Molar                                                  conjugate    Yield (%) Ratios   (% Orig. Binding)                             ______________________________________                                        Semicarbazone                                                                              83        3.42     97                                            Carbazone    74        4.37     92                                            Thiosemicarbazone                                                                          61        2.51     72                                            Carboxylate- 78        2.35     97                                            Hydrazone                                                                     Arylhydrazone                                                                              86        2.52     91                                            ______________________________________                                    

As shown in Table 1, the 5E9 immunoconjugates retained over 90% (exceptfor thiosemicarbazone) of the original binding activity of theunconjugated 5E9. This demonstrates that the conjugation of the ADMderivatives to the 5E9 MAb resulted in the loss of relatively smalldegrees of antibody binding activity. The protein yields indicate thathigh amounts of protein were retained throughout the conjugationprocedure.

Release of ADM From The Carbazone Immunoconjugate

The release rates of ADM from the carbazone immunoconjugate of theinvention at pH 4.5, 5.0 and 7.4 were also studied by HPLC analysis asdescribed above in Example 6 for the ADM-derivatives of the invention.As shown in FIG. 13, the release rate of ADM from the immunoconjugate isessentially the same as that shown for the carbazone derivative of ADMin Example 6. The amount of material released from theADM-immunoconjugate increased as the pH was lowered from 7 to 4. ThisADM-immunoconjugate has an acidsensitive linkage group which results inrelease of ADM from the antibody protein. These results are consistentwith the existence of a hydrazone bond joining the ADM to thebifunctional compound.

The experimental data described herein demonstrate that an ADM moiety isreleased from the immunoconjugates of this invention under "physiologic"conditions, i.e. acidic conditions typical of the lysosomal environment.

Cytotoxic Activity of the Immunoconjugates

The cytotoxicity of the immunoconjugates of the invention was determinedby in vitro testing using the Colony Formation Assay in soft agar usingDaudi (Burkitt's lymphoma) cells (phenotype: 5E9 , ATCC No. HB21) asdescribed by Greenfield et al., European Patent Application No. 328,147,supra. The Daudi cells were grown in complete medium (RPMI 1640 mediumplus 10% fetal calf serum (FCS)). 1×10⁵ cells in 1 ml of medium wereexposed for 1.5 hours to serially diluted 5E9-ADM immunoconjugates orunconjugated ADM. Triplicate determinations were done for each dilution.Controls consisted of similarly treated cells not exposed to drugs. Thecells were then washed and suspended in RPMI 1640 medium containing 15%FBS and 0.3% agarose (Marine Colloid, Rockland, Me.). One ml of the cellsuspension (1×10³) cells was then overlayed onto a 0.4% agarose layer in6 well microtiter plates (Costar, Cambridge, Mass.). Samples wereincubated for 7-10 days at 37° C. and the resulting colonies werestained with 0.5 ml of 1 mg/ml of p-iodonitrotetrazolium violet (SigmaChemical Co., St. Louis, Mo.) for 48 hours. Colonies were counted usingan Optimax 40-10 image analyzer and the inhibition of colony formationwas determined by comparing drug-treated or immunoconjugate-treatedcells to the untreated control. The results are presented in Table 2,below, as IC₅₀ (the concentration required to inhibit colony formationby 50%).

                  TABLE 2                                                         ______________________________________                                        5E9 Immunoconjugate   IC.sub.50 (M).sup.a                                     ______________________________________                                        Semicarbazone         >5.1 × 10.sup.-7                                  Carbazone              4.0 × 10.sup.-7                                  Thiosemicarbazone      3.0 × 10.sup.-7                                  Carboxylatehydrazone   5.9 × 10.sup.-7                                  Arylhydrazone         >6.4 × 10.sup.-7                                  ______________________________________                                         .sup.a (M) = molar concentration of immunoconjugate required to inhibit       colony formation by 50% measured after 24 h.                             

As shown in Table 2, in addition to releasing ADM, all of thepH-sensitive immunoconjugates possess significant cytotoxic activity invitro.

EXAMPLE 9 Preparation of an Anthracycline Immunoconjugate containing aThioether Linkage

This example describes an alternative embodiment for the preparation ofan anthracycline immunoconjugate according to the present invention,wherein ADM is conjugated to a monoclonal antibody via one of theADM-derivatives of the invention, prepared as described above inexamples 1-5, and having any of the five bonds: semicarbazone,carbazone, thiosemicarbazone, carboxylatehydrazone and arylhydrazone asits site of attachment to the ADM molecule. Additionally, theimmunoconjugate has a thioether linkage as part of its attachment to theantibody.

MAb 5E9 (2.5 mg in 2.5 ml of PBS) is reacted with SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate 59.5 μg in 100 μltetrahydrofuran) at 30° C. for 30 min. The pH is adjusted to 6.0 withsodium citrate buffer. The mixture is passed through a PD-10 gelfiltration column (Pharmacia) to separate maleimide-containing antibodyfrom unreacted materials. The ADM-derivatives (1 mg), prepared asdescribed above, are then dissolved in 1 ml MeOH/H₂ O (9:1) and 0.5μmoles of each of the ADM-derivatives is reacted with 0.5 μmoles oftri-n-butylphosphine in 4:1 acetone:H₂ O to prepare the reduced form ofthe ADM derivative. After 10 min, 0.1 M sulfur in toulene is added todestroy remaining phosphine. The reduced ADM-derivatives are then mixedwith the 5E9 maleimide-containing MAb. Immunoconjugates so produced arepurified by passage through a PD-10 gel filtration column. In caseswhere removal of toluene solvent is not complete and an organic solventlayer separates floating some protein from the reaction mixture, agentle stream of air is used to remove the solvent and the denaturedprotein is removed by spinning the mixture for 2 min at 16,000×g. Theclear supernatant contains the immunoconjugates and is gel filtered andanalyzed in PBS at pH 7.4. The ADM/antibody molar ratio is determinedspectrophotometrically using OD₂₈₀ and OD₄₉₅ as described above. Atypical reaction yields immunoconjugates with molar ratios between 3 and4.

The binding and cytotoxic activity of immunoconjugates prepared asdescribed in this example are tested as described above.

The above examples demonstrate the preparation of novel N-substitutedhydrazine bifunctional compounds, novel N-substituted hydrazonederivatives of ADM made with these bifunctional compounds and novelimmunoconjugates in which ADM was conjugated to an antibody via a novelacid-sensitive linkage. The bifunctional compounds were readilyconjugated with a cytotoxic reagent, ADM and a cell targeting molecule,a monoclonal antibody. The conjugates retained both antibody bindingactivity, (i.e. target cell specificity) and cytotoxic drug activity andreleased free, unmodified ADM under acidic conditions typical of thecellular environment of target cells.

Thus, the novel bifunctional compounds of the invention immunoconjugatesof the invention show promise for conjugating useful molecules,particularly for delivering cytotoxic drugs to a target cell populationfor the preferential killing of those cells in the treatment of diseasessuch as cancers and other tumors, non-cytocidal viral or otherpathogenic infections and autoimmune disorders.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the bifunctional compounds,derivatives of cytotoxic reagents, conjugates and methods of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the claims appended hereto rather than bythe specific embodiments which have been presented hereinbefore by wayof example.

We claim:
 1. A bifunctional N-substituted hydrazine compound having theformula: ps

    H.sub.2 NNHCONH(CH.sub.2).sub.n SSR.sup.8

wherein n is an integer from 1 to 10; and ##STR7## wherein X is H, NO₂or halogen.
 2. N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide. 3.A N-substituted hydrazine bifunctional compound having the formula:

    H.sub.2 NNHCONHNHCONH(CH.sub.2).sub.n SSR.sup.8

wherein n is an integer from 1 to 10; and ##STR8## wherein X is h, NO₂or halogen.
 4. 2-[[[2-[(2-pyridinyl)dithio]ethyl]amino]carbonyl]carbonicdihydrazide.
 5. A N-substituted hydrazine bifunctional compound havingthe formula:

    H.sub.2 NNHCSNH(CH.sub.2).sub.m CH═CH(CH.sub.2).sub.n SSR.sup.8

wherein m, n are integers from 1 to 10, which are the same or different;and ##STR9## wherein X is H, NO₂ or halogen. 6.N-4-[(2-pyridinyl)dithio-2-butenyl]hydrazinecarbothioamide.
 7. AN-substituted hydrazine bifunctional compound having the formula:

    H.sub.2 NNHCOO(CH.sub.2).sub.n SSR.sup.8

wherein n is an integer from 1 to 10; and ##STR10## wherein X is H, NO₂or halogen.
 8. 2[(2-pyridinyl)dithio]ethyl hydrazinecarboxylate.
 9. AN-substituted hydrazine bifunctional compound having the formula:

    H.sub.2 NNH-Ar-CONH(CH.sub.2).sub.n SSR.sup.8

wherein n is an integer from 1 to 10; and ##STR11## wherein X is H, NO₂or halogen and Ar is ##STR12## 10.N-[2-[(2-pyridinyl)dithio)ethyl]-4-hydrazinobenzamide.
 11. Ananthracycline derivative prepared by reacting the compound of claim 1,3, 5, 7 or 9 with an anthracycline.
 12. The anthracycline derivative ofclaim 11 wherein said anthracycline is selected from the groupconsisting of adriamycin, daunomycin, detorubicin, carminomycin,idarubicin, epirubicin, esorubicin, 4'-THP-adriamycin, AD-32 and3'-deamino-3'-(3-cyano-4-morpholinyl)-doxorubicin.
 13. Adriamycin13-N-[2-[(2-pyridinyl)dithio]ethyl]hydrazinecarboxamide semicarbazonehydrochloride.
 14. Adriamycin13-2-[[[2-[(2-pyridinyl)dithio]ethyl]amino] carbonyl]carbonicdihydrazide carbazone hydrochloride.
 15. Adriamycin13-N-4-[(2-pyridinyl)dithio]-2-butenylhydrazinecarbothioamidethiosemicarbazone hydrochloride.
 16. Adriamycin13-2-[(2-pyridinyl)dithio]ethyl hydrazinecarboxylatecarboxylatehydrazone hydrochloride.
 17. Adriamycin13-N-[2-[(2-pyridinyldithio)ethyl]-4-hydrazinobenzamide arylhydrazonehydrochloride.
 18. An anthracycline derivative having the formula:##STR13## wherein: R¹ is NHCONH(CH₂)_(n) SSR₈ ; NHCONHNHCONH(CH₂)_(n)SSR₈ ;NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) SSR₈ ; NHCOO(CH₂)_(n) SSR₈ ;NH--Ar--CONH(CH₂)_(n) SSR₈ ; NCONH(CH₂)_(n) S--H; NHCONHNHCONH(CH₂)_(n)S--H;NHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) S--H; HNCOO(CH₂)_(n) S--H orNH--Ar--CONH(CH₂)_(n) S--H wherein m, n are integers from 1 to 10, whichare the same or different; ##STR14## wherein X is H, NO₂ or halogen andAr is ##STR15## R₂ is CH₃, HC₂ OH, CH₂ OCO(CH₂)₃ CH₃, or CH₂ OCOCH(OC₂H₅)₂ ; R₃ is OCH₃, OH or hydrogen; R₄ is NH₂, NHCOCF₃, 4-morpholinyl,3-cyano-4-morpholinyl, 1-piperidinyl, 4-methoxy-1-piperdinyl, benzylamine, dibenzyl amine, cyanomethyl amine or 1-cyano-2-methoxyethylamine; R₅ is OH, O-THP or hydrogen; and R₆ is OH or hydrogen, providedthat R₆ is not OH when R₅ is OH or O-THP.
 19. An anthracyclinederivative having the formula: ##STR16## wherein: R₁ is NHCONH(CH₂)_(n)SSR₈ ; NHCONHNHCONH(CH₂)_(n) SSR₈ ;NCHSNH(CH₂)_(m) CH═CH(CH₂)_(n) SSR₈ ;NHCOO(CH₂)_(n) SSR₈ ; NH-Ar-CONH(CH₂)_(n) SSR₈ ; NCONH(CH₂)_(n) S--H;NHCONHNHCONH(CH₂)_(n) S--H; NNHCSNH(CH₂)_(m) CH═CH(CH₂)_(n) S--H;NHCOO(CH₂)_(n) S--H or NH-Ar-CONH(CH₂)_(n) S--H wherein m, n areintegers from 1 to 10, which are the same or different; ##STR17##wherein X is H, NO₂ or halogen and Ar is ##STR18## R₂ is CH₃, CH₂ OH,CH₂ OCO(CH₂)₃ CH₃, or CH₂ OCOCH(OC₂ H₅)₂ ; R₃ is OCH₃, OH or hydrogen;R₄ and R₇ are independently hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, aralkyl orsubstituted aralkyl; or R₄, R₇ and N together form a 4-7 membered ring,wherein said ring may be optionally substituted; R₅ is OH, O-THP orhydrogen; and R₆ is OH or hydrogen, provided that R₆ is not OH when R₅is OH or O-THP.
 20. A conjugate comprising the anthracycline derivativeof claim 18 or 19 conjugated to at least one molecule reactive with atarget cell population.
 21. A pharmaceutically acceptable compositionuseful in the treatment of disease which comprises a pharmaceuticallyeffective amount of at least one conjugate according to claim 20 and apharmaceutically acceptable carrier.